Cyclic hydrocarbons with an aminoalkyl sidechain

ABSTRACT

Provided are cyclic hydrocarbons of Formula I ##STR1## with an aminoalkyl sidechain that are useful for treating phospholipase A2 mediated conditions, diabetes, and obesity.

FIELD OF INVENTION

This invention relates to novel compositions of matter. Moreparticularly, the invention relates to cyclic hydrocarbons with anaminoalkyl sidechain that are useful for inhibiting phospholipase A2 andfor treating diabetes and obesity.

INFORMATION DISCLOSURE

The important role of phospholipase A2 in mammalian metabolism throughthe formation of prostaglandins is now well known. See W. Vogt, Advancesin Prostaglandins and Thromboxane Research, 3, p. 89 (1978); P. C.Isakson, et al., Advances in Prostaglandin and Thromboxane Research, 3,page 113, (1978). Phospholipase A2 is responsible for the hydrolysis ofarachidonic acid-containing phospholipids, thereby providing substratefor the multiple enzymes of the arachidonic acid cascade.

The products of the arachidonic acid cascade are varied. These productsinclude prostaglandins, thromboxanes, leukotrienes, and otherhydroxylated derivatives of arachidonic acid. All of the foregoing arereferred to as "eicosanoids." While generally the products of thecascade are beneficial, in certain disease processes and otherconditions the excessive production of eicosanoids induces deleteriousconsequences such as inflammation (see paper by N. A. Plummer, et al.;abstracted in Journal of Investigative Dermatology, 68, p. 246 (1977));erythema (N. A. Plummer, supra); platelet aggregation (B. B. Vargafting,J. Pharm. Pharmacol., 29, pp. 222-228 (1977)); and the release of SRS-A(slow reacting substance-anaphylaxis), a known mediator of allergicresponses. The inhibition of phospholipase A2 prevents these and similarconditions mediated by the action of this enzyme.

Some inhibitors of phospholipase A2 are known. R. J. Flower and G. J.Blackwell have shown that certain anti-inflammatory steroids inducebiosynthesis of a phospholipase A2 inhibitor which preventsprostaglandin generation. See Nature, 278, p. 456 (1979). These steroidsare not direct inhibitors of phospholipase A2, but rather stimulate thesynthesis of a phospholipase inhibiting factor called lipocortin,lipomodulin, or macrocortin.

Some examples of direct phospholipase A2 inhibition are known.Indomethacin, a drug with anti-inflammatory properties, has been shownto inhibit phospholipase A2 enzymes. See K. L. Kaplan, et al., Proc.Natl. Acad. Sci., 75, pp. 2955-2988 (1978).

Indomethacin has been shown to inhibit phospholipase A2 enzymes,isolated respectively from the venoms of Russell's Viper, Crotalusadamanteus, and bee, and from pig pancreas. Certain local anestheticshave been shown to inhibit phospholipase A2 activity by competing withcalcium ion, which appears to be a requirement for phospholipaseactivity. See W. Vogt, Advances in Prostaglandin and ThromboxaneResearch, 3, p. 89 (1978) and E. Vallee et al., J. Pharm. Pharmacol.,31, pp. 588-92 (1974). Bromphenacyl bromide has been shown to inhibitphospholipase A2 by acylating a histidine residue which is at the activesite of the enzyme. See M. Roberts, et al., J. of Biol. Chem., 252, pp.2405-2411 (1977). R. Blackwell, et al., British J. Pharmacy, 62, p.79-89 (1978) has disclosed that mepacrine inhibits the activity ofphospholipase A2 derived from perfused guniea pig lung. Certainbutyrophenones are disclosed as phospholipase A2 inhibitors in U.S. Pat.No. 4,239,780. D. P. Wallach and V. J. R. Brown, Bioch. Pharmacol., 30,pp. 1315-24 (1981) also refer to several compounds that inhibitphospholipase A2.

Some of the steroids employed for synthesizing compounds of the presentinvention and useful in some of the methods of treatment are known. Seethe doctoral thesis, L. J. Griggs, "Part I. Synthetic Approaches to 5-and 16-Thiaestrone. Part II. Estrone with a Diazacholesterol SideChain," University of Michigan (1965). These compounds are statedtherein to be potential hypocholesterolemic agents. U.S. Pat. No.3,370,070 discloses similar steroid compounds which are useful ashypocholesterolemic agents and as antibacterial, anti-protozoal, andanti-algal agents.

Some of the steroidal compounds herein are also referred to in U.S. Pat.No. 3,284,475 and in P. D. Klimstra, et al., "HypocholesterolemicAgents. VI. A- and B-Ring-Modified Azacholesterols", J. Med. Chem., 9,pp. 323-326 (1966).

The present invention also relates to antidiabetic agents. Hyperglycemiarefers to a condition commonly found in patients suffering frommature-onset diabetes mellitus and other diseases in which impairment ofpancreatic function is a consequence thereof. Accordingly,hyper-glycemic patients are those exhibiting elevated serum glucoselevels. Failure to adequately control such elevated serum glucose levelshas been assoicated in such patients with untoward cardiovasculareffects (myocardioischemia, stroke, and peripheral vascular diseases),lethargy, coma, blindness, kidney failure and even death.

While conventional treatment for these hyperglycemic conditions mayinclude diet (e.g. restriction of carbohydrate intake) and insulininjection, one important means of treating such patients is with oralantidiabetic agents such as those disclosed herein.

SUMMARY OF THE INVENTION

The present invention relates to cyclic hydrocarbons of formula Iwherein:

A compound of the formula ##STR2## wherein; ##STR3## 1. wherein ##STR4##(1) wherein D is (a) H, (b) CH₃, or (c) no bond;

(2) wherein E and J are

(a) H, (b) R₅ O--, or (c) --N(CH₃)--(CH₂)₃ --N(CH₃)₂ with the provisosthat when E is H, the 5,6 bond is saturated and that when J is H, X₂ andX₃ are H;

(3) wherein G is

(a) nothing, or (b) →O;

(4) wherein R₅ is

(a) H, (b) C1-C3 alkyl, (c) benzyl, (d) acyl, (e) C(O)H, (f) HOCH₂CH(OH)CH₂, (g) R₄ --OC(O)CH₂ ;

(5) wherein X₂ and X₃ are

(a) H, (b) NO₂, (c) NH₂, (d) OH, or (e) halogen;

B. C8-C20 cycloalkyl, C. 2- or 4-cyclohexylcyclohexyl, D.4-bicyclohexylcyclohexyl, E. 4-bicyclohexenylcyclohexyl, F.3-cyclopentylcyclopentyl, G. 1-, 3- or4-(2-decahydronaphthyl)cyclohexyl, H. 1- or 2-tetradecahydroanthracenyl,I. 2- or 3-tetradecahydrophenanthrenyl, J. 1- or2-dodecahydro-1H-phenallyl, K. 1- or 2 hexadecahydropyrenyl, L. 1- or2-octadecahydrotriphenylenyl, M. 1- or 2-octadecahydrochrysenyl, N. 1-or 2-octadecahydronaphthacenyl, O. phenylcyclohexyl, cyclohexyl, P.adamantyl, Q. pyrenyl, R. 3-fluorobiphenylyl, or S. 1- or 2- decalinyl;

II. wherein X₁ is

A. NR₁, B. NR₁ R₁₃, C. N⁺ ·R₁ ·R₁ ·R₁₃ X⁻, or D. --O--C(O)--CH-- ((CH₂)₃--NH₂)(NH₂);

1. wherein X⁻ is a pharmaceutically acceptable anion;

2. wherein R₁₃ is

a. methyl, or b. →O;

3. wherein R₁ is

a. H, b. --CHO, c. --COCH₃, d. C1-C6 alkyl, e. --(CH₂)_(r) --CO₂ R₄, f.--CH₂ CH═CH₂, g. --(CH₂)_(p) --X₄, h. --(CH₂)_(m) -- N(R₆)(R₇), i.--(CH₂)_(p) --O-- (CH₂)_(p) --N(R₆)(R₇), j. --(CH₂)_(p)--Y--C(═NH)--NH₂, k. --(CH₂)_(q) --CH(NH₂)--COOR₁₆, l. --(CH₂)_(p)--N═C(R₁₄)(R₁₅), m. --(CH₂)_(p) --NH--C(CH₃)₂ --CH₂ --C(O)--CH₃,##STR5## (1) wherein R₄ is (a) H, or (b) C1-C2 alkyl;

(2) wherein X₄ is

(a) OH, (b) OCH₃, (c) OC₂ H₅, (d) OCH₂ CH₂ OH, (e) OTs, (f) OMs, (g) Cl,(h) Br, (j) aziridinyl, or ##STR6## (i) wherein R₁₂ is (ii)(a) C1-C2alkyl, (ii)(b) benzyl, (ii)(c) CH₂ Cl, (ii)(d) →O, (ii)(e) CH₂ COOC₂ H₅,or (ii)(f) C3-C18 straight chain alkyl;

(3) wherein R₆ is

(a) H, (b) C1-C13 alkyl, (c) benzyl, (d) phenyl, (e) --(CH₂)_(p)-N(R₁₀)(R₁₁), (f) C(O)CH₃, ##STR7## (i) wherein R₁₀ and R₁₁ are (i)(a)H, (i)(b) C1-C2 alkyl, or (i)(c) (CH₂)₃ --NH₂ ;

(ii) wherein R₁₀ and R₁₁ together are

(ii)(a) ##STR8## (4) wherein R₇ is (a) H, (b) C1-C2 alkyl, (c)--(CH₂)_(p) --N(R₁₀)(R₁₁), or (d) CHO;

(5) wherein R₆ and R₇ together are ##STR9## (i) wherein X₆ is (i)wherein X₆ is

(i)(a) O, (i)(b) NH, (i)(c) NCH₃, or (i)(d) N(CH₂)_(q) NH₂ ; ##STR10##(6) wherein Y is (a) NH, or (b) S;

(7) wherein R₈ is

(a) H, (b) C1-C2 alkyl, (c) OCH₃, (d) NO₂, (e)NH₂, (f) NHCOCH₃, (g) CN,(h) CH₂ NH₂, (i) CONH₂, (j) Cl, (k) Br, or (l) COOCH₃ ;

(8) wherein R₉ is

(a) H, (b) methyl, (c) benzyl, or (d) --(CH₂)_(p) N(R₁₀)(R₁₁);

(9) wherein R₁₄ is

(a) H, or (b) C1-C6 alkyl;

(10) wherein R₁₅ is C1-C6 alkyl;

(11) wherein R₁₆ is

(a) H, or

(b) C1-C4 alkyl;

(12) wherein X₇ and X₈ are the same or different and are

(a) H, (b) CH₃, (c) CF₃, (d) halogen, (e) OH, (f) OCH₃, (g) NO₂, (h)NH₂, (i) NHR₄, (j) NR₄ R₄, (k) --CH₂ NH₂, (l) --CH₂ NHR₂, (m) --SO₂N(R₃)(R₄), (n) --CO₂ R₄, (o) CON(R₃)(R₄), (p) CH₂ N(R₃)--(R₄), or (q)tetrazolyl;

III. wherein R₂ is

A. H, B. C1-C4 alkyl, C. benzyl, D. --(CH₂)_(p) --N(R₆)(R₇), E.##STR11## N(→O)(R₆)(R₇), H. --(CH₂)_(p) N⁺ (CH₂ --Ph)(R₆)(R₇), or I.--(CH₂)_(p) N⁺ (CH₃)(R₆)(R₇), J. nothing;

IV. wherein R₁ and R₂ together are ##STR12## 1. wherein X₅ is a. O, b.NH, c. NCH₃, or d. S; ##STR13## 2. wherein X₉ is a. O, b. NH, or c. NCH₃;

V. wherein R₃ is

A. H, B. C1-C2 alkyl, or C. CH₂ OH;

wherein m is 2-8;

wherein n is 0-1;

wherein p is 2-8;

wherein q is 2-4;

wherein r is 1-8;

wherein s is 2-8; and

pharmacologically acceptable salts thereof;

with the proviso that when n is 1 and R₁ is --(CH₂)_(m) --N(R₆)(R₇)wherein m is 2 or 3 and R₂ is H or CH₃, or when R₂ is --(CH₂)_(m)--N(R₆)--(R7) wherein m is 2 or 3 and R₁ is H, CH₃, CHO, or CH₃ CO, thenR₆ and R₇ cannot both be hydrogen, methyl, or ethyl;

and with the proviso that when n is 0 and R₁ is --(CH₂)_(m)--N(R₆)--(R7) wherein m is 2 or 3 and R₂ is H or CH₃, or when R₂ is--(CH₂)_(m) -- --N(R₆)(R7) wherein m is 2 or 3 and R₁ is H, CH₃, CHO, orCH₃ CO, then R₆ and R₇ cannot both be hydrogen, methyl, or ethyl,propyl, or isopropyl;

and with the proviso that when n is 0 and one of R₁ and R₂ is--(CH₂)_(m) --N(R₆)(7) wherein m is 3, and the other is H or methyl,then R₆ and R₇ cannot be H or methyl;

and with the proviso that when n is 0 and X1 is NR₁, then R₁ cannot beCHO when R₂ is H;

for each of the foregoing provisos, Z is ##STR14##

The invention also relates to methods of treatment of phospholipase A2mediated conditions (PMC), and methods of treatment of diabetes, obesityand atherosclerosis, employing the compounds and methods of theinvention.

Compounds of the present invention have been tested in standardlaboratory tests to evaluate their ability to inhibit phospholipase A2.In the pefused guinea pig lung,N[3-(dimethylamino)propyl]-N-methyl-3-methoxyestra-2,5(10)-dien-17b-aminehas been shown to be preferred, exhibiting complete inhibition of theenzyme at 4×10-7 Molar.

Thus, some of the compounds of the present invention are useful wheneverit is medically necessary or desirable to inhibit phospholipase A2 in amammalian systems. They are particularly useful in treating symptoms orconditions resulting from the action of the arachidonic acid cascade.

The symptoms or conditions treated or prevented by the phospholipase A2inhibitory compounds of this invention are those which are produced as aresult of the excessive stimulation of the arachidonic acid cascadeduring certain disease processes or conditions. The multiple enzymes ofthe cascade act upon 5,8,11,14-eicosatetraenoic acid to produceprostaglandins, leukotrienes, and hydroxylated derivatives. At certaintimes during these disease processes or conditions, some of theseproducts are responsible for the symptoms or conditions noted above,e.g., inflammation, erythema, allergic responses, and similarconditions. Phospholipase A2 provides the substrate for these enzymes ofthe cascade by hydrolysis of arachidonate-rich phospholipids. Thus,phospholipase A2 is an important mediator in these conditions.Inhibition of this enzyme by the method of this invention is thuseffective to treat or prevent the symptoms or conditions, which aredesignated as PMC's (phospholipase A2 mediated conditions).

The precise mechanisms of the disease processes or conditions whichstimulate the arachidonic acid cascade are not clearly understood. Theessential prerequisite, however, is enhanced activity of thephospholipases which provide arachidonate to the series of reactionsdesignated as the arachidonic acid cascade. The method of this inventionis simply to block the action of the phospholipases and cut off the flowof arachidonate into the cascade, irrespective of the stimulus orstimuli which may be present. This is accomplished with thephospholipase A2 inhibitory compounds of the present invention. Thus,the method of phospholipase A2 inhibition of this invention is suitablefor treating seemingly unrelated diseases whose common element is thestimulation of the arachidonic acid cascade. PMC includes all untowardconditions or symptoms which are the result of the excessive stimulationof the arachidonic acid cascade. These conditions encompass allergicdiseases, inflammatory conditions (including chronic inflammatoryconditions such as rheumatoid arthritis), burns, and hypoxic conditionsat the cellular level such as coronary infarcts, or infarcts of othertissues. In these latter infarct conditions it is desirable to blockphospholipase A2 activity to prevent the destruction of thephospholipids that are substrates for phospholipases which also areintegral structural components of cellular membranes.

The phospholipase A2 inhibition method of this invention is used on anymammal whose metabolic system includes the phospholipase inducedarachidonic acid cascade. The mammals which are preferred are generallydomesticated animals and humans. Humans are the most preferred mammalsto be treated by the method of this invention.

The dosage regimen for preventing or treating PMC by the compounds ofthis invention is selected in accordance with a variety of factors.,including the type, age, weight, sex and medical condition of themammal, the severity of the PMC and the particular compound employed. Anordinarily skilled physician or veterinarian will readily determine andprescribe the effective amount of the anti-PMC agent to prevent orarrest the progress of the condition. In so preceeding the physician orveterinarian could employ relatively low dosages at first, subsequentlyincreasing the dose until a desired or maximum response is obtained.

Initial dosages of the compounds of this invention can be from about0.003 to 3.0 g per 70 kg mammal per 6-8 hours orally. When other formsof administration are employed, equivalent doses are administered. Whendosages beyond 45 mg/kg employed, care should be taken with eachsubsequent dose to monitor possible toxic effects.

The degradation of cell membranes by phospholipase A2, hydrolyzing thephospholipid components of the membrane, is believed to be a componentin the cellular death resulting from hypoxic states such as coronaryinfarcts, ligation of the aorta during surgery for aortic aneurysms(resulting in kidney damage), and the like . Inhibition of phospholipaseA2 by these compounds could greatly ameliorate the cellular damageresulting from such causes. See Zalewski, et al., Clinical Research 31,p. 227 (1983). This is a preferred use of these compounds.

Asthma is a disease of the lungs in which a wide variety of stimuli canresult in an asthmatic attack. These stimuli range from damp cold air toallergens in the environment. The asthmatic response is characterized byconstriction of the bronchioles leading to increased airway resistance.There is an early constrictive phase due to histamine release from mastcells, as well as other modulators, e.g., peptides. A late sustainedphase then occurs which in human beings may reach a maximum in 6-8hours. This phase is slower in onset and disappearance, and is due to acomplex of products of the arachidonic acid cascade. These productsinclude thromboxanes, prostaglandins, and leukotrienes. The precursorfor all of these eicosanoids is arachidonate which is released fromesterified forms in membranes to the appropriate enzymes by the actionof phospholipase A2. See, e.g., "Corticosteroid Treatment in AllergicAirway Diseases," Proceedings of a Symposium in Copenhagen Oct. 1-2,1981 (Editiors: T. H. Clark, N. Myginfd, and O. Selroos,Munksgaard/Copenhagen 1982). Thus, a block of phospholipase A2, which isphysiologically acceptable, will prevent release of eicosanoids in thelung thought to be responsible for the "2nd wave" of airway resistance.This is a preferred use of some of the compounds of this invention.

Thus, the phospholipase A2 inhibitory compounds are useful in thetreatment of asthma. For example, these compounds are useful asbronchodilators or as inhibitors of mediators such as SRS-A which arereleased from cells activated by an antigen-antibody complex. Further,these compounds control spasm and facilitate breathing in conditionssuch as bronchial asthma, bronchitis, bronchiectasis, pneumonia andemphysema. For these purposes, these compounds are administered in avariety of dosage forms, e.g., orally in the form of tablets, capsules,or liquids; rectally in the form of suppositories; parenterally,subcutaneously, intradermally, or intramuscularly, with intravenousadministration being preferred in emergency situations, by inhalation inthe form of aerosols or solutions for nebulizers; or by insufflation inthe form of powder. These compounds are effectively administered tohuman asthma patients by oral inhalation or by aerosol inhalation.

Doses in the range of about 0.01 to 50 mg per kg of body weight are used1 to 4 times a day, the exact dose depending on the age, weight, andcondition of the patient and on the frequency and route ofadministration. For the above use these compounds can be combinedadvantageously with other anti-asthmatic agents, such assympathomimetics (isoproterenol, phenylephrine, ephedrine, etc.);xanthine derivatives (theophylline and aminophylline); andcorticosteroids (e.g., prednisolone).

For administration by the oral inhalation route with conventionalnebulizers or by oxygen aerosolization it is convenient to provide theinstant active ingredient in dilute solution, preferably atconcentrations of about 1 part of medicament to form about 100 to 200parts of weight of total solution. Entirely conventional additives maybe employed to stabilize these solutions or to provide isotonic media,for example, sodium chloride, sodium citrate, citric acid, sodiumbisulfite, and the like can be employed.

A self-propelled dosage unit suitable for inhalation therapy foradministering the active ingredient in aerosol form comprises the activeingredient suspended in an inert propellant (such as a mixture ofdichlorodifluoromethane and dichlorotetrafluoroethane) together with aco-solvent, such as ethanol, flavoring materials and stabilizers.Instead of a co-solvent there can also be used a dispensing agent suchas oleyl alcohol. Suitable means to employ the aerosol inhalationtherapy technique are described fully in U.S. Pat. No. 2,868,691, forexample.

These novel compounds are useful as anti-inflammatory agents in mammalsand especially humans, and for this purpose, are administeredsystemically and preferably orally. For oral administration, a doserange of 0.05 to 50 mg per kg of human body weight is used to giverelief from pain associated with inflammatory disorders such asrheumatoid arthritis. They are also administered intravenously inaggravated cases of inflammation, preferably in a dose range 0.01 to 100g per kg per min until relief from pain is attained. When used for thesepurposes, these novel compounds cause fewer and lesser undesirable sideeffects than do known synthetase inhibitors used to treat inflammation,for example, aspirin and indomethacin. When these novel compounds areadministered orally, they are formulated as tablets, capsules, or asliquid preparations, with the usual pharmaceutical carriers, binders,and the like. For intravenous use, sterile isotonic solutions arepreferred.

These compounds are useful whenever it is desired to inhibit plateletaggregation, reduce the adhesive character of platelets, and remove orprevent the formation of thrombi in mammals, including man, rabbits,dogs, and rats. For example, these compounds are useful in theprevention of myocardial infarcts, to prevent post-operative thrombosis,to promote patency of vascular grafts follwoing surgery, and to treatconditions such as atherosclerosis, arteriosclerosis, blood clottingdefects due to lipemia, and other clinical conditions. For thesepurposes, these compounds are administered systemically, e.g.,intravenously, subcutaneously, intramuscularly, and in the form ofsterile implants for prolonged action. For rapid response especially inemergency situations, the intravenous route of administration ispreferred. Doses in the range about 0.005 to about 20 mg per kg of bodyweight per day are used, are exact dose depending on the age, weight,and condition of the patient or animal, and on the frequency and routeof administration.

Increased phospholipase activity has been observed after central nervoussystem (CNS) trauma, e.g., brain and spinal cord injury. See, E. P. Wei,et al., J. Neurosurg., 56, pp. 695-698 (1982) and E. D. Hall and J. M.Braughler, Surgical Neurology, 18, pp. 320-327 (1982). Thus,phospholipase inhibitors, such as the compounds of the presentinvention, would be useful in the treatment or prevention of suchconditions.

The method of this invention is useful both in treating a PMC or symptomwhich has already manifested itself in the mammal as well as theprevention of these conditions or symptoms in mammals including thoseparticularly susceptible to them. Employment of the method of thisinvention prior to the developement of a PMC would prevent the formationof the prostaglandins and similar products necessary for suchconditions. Thus, the method of this invention can be used to preventedema and erythema from sunburn by administering these compounds priorto exposure to sunlight. The compounds of this invention could beadministered to persons suffering from hayfever or similar allergiesprior to exposure to the allergenic substances to which hayfeversufferers are sensitive. In a like manner, a physician or veterinariancould readily determine other mammals or persons susceptible to a PMC.

It is most preferred to use the compounds of this invention in thetreatment or prevention of asthma and in the treatment or prevention ofcellular death resulting from hypoxic states.

The actual inhibition of phospholipase A2 by the method of thisinvention takes place on a cellular level. Administration of thephospholipase A2 inhibitory compounds of this invention can thus be byany manner that will allow for phospholipase A2 inhibition in theaffected tissues or organs. The preferred route in most cases is tosystemically administer the compounds, i.e., to allow them to enter themammal's bloodstream and thus be distributed throughout the mammal'ssystem. In certain cases, where the PMC is of a localized nature (e.g.,sunburn or psoriasis), topical administration (e.g., transdermal) may beemployed in order that the phospholipase A2 inhibition is confined tothe aflicted area.

Since the diseases or conditions resulting from the arachidonic acidcascade are varied, methods of administering these compounds must dependof the particular PMC to be treated. Regardless of the route ofadministration selected the compounds used in the process of the presentinvention are formulated into pharmaceutically acceptable dosage formsby conventional methods known to the pharmaceutical art.

Thus, the compounds can be administered orally in forms such as pills,capsules, solutions or suspensions. They may also be administeredrectally or vaginally in forms such as suppositories or bougies. Theymay also be introduced parenterally, e.g., subcutaneously,intravenously, or intramuscularly using sterile injectable forms knownto the pharmaceutical art. For treatment of conditions such as erythemathe compounds of this invention may also be administered topically inthe form of ointments, creams, gels, or the like.

The compounds of the present invention may be formulated intopharmaceutical compositions, employing a pharmaceutically acceptablecarrier.

The pharmaceutical forms contemplated by this invention includepharmaceutical compositions suitable for oral, parenteral, vaginal,topical, and rectal use, e.g., tablets, powder packets, chachets,dragees, suppositories, bougies, and the like. Suitable diluents orcarriers such as carbohydrates (lactose), proteins, lipids, calciumphosphate, cornstarch, stearic acid, methylcellulose and the like may beused as carriers or for coating purposes. Oil, e.g., coconut oil, sesameoil, safflower oil, cottonseed oil, peanut oil may be used for preparingsolutions or suspensions of the active drug. Sweetening, coloring andflavoring agents may be added. By "pharmaceutical excipient" is meantany of these and similar well known forms of drug formulations.

In general the preferred route of administration depends on thecondition being treated. For asthma, oral inhalation or aerosolinhalation is preferred. For most other conditions the preferred mode ofadministration is oral.

Some of the compounds of the present invention are useful ashypoglycemic agents in non-insulin dependent diabetes mellitus (NIDDM)with insulin resistance. NIDDM refers to a condition commonly found inpatients suffering from elevated serum glucose levels resulting from animpairment of tissue response to insulin and/or an impairment ofpancretic islet function. Failure to adequately control such elevatedserum glucose levels (hyperglycemia) has been associated in suchpatients with untoward cardiovascular effects (myocardioischemia,stroke, and peripheral vascular diseases), lethargy, coma, and evendeath.

While conventional treatment for these hyperglycemic conditions mayinclude diet (e.g., restriction of carbohydrate intake) and insulininjection, one important means of treating such patients is with oralhypoglycemic agents.

Some of the compounds of this inventon have the ability to lower theserum glucose levels in KKA^(y) mice with spontaneous diabetes, and theyare expected to be of value in the treatment of NIDDM and itscomplications in mammals, including human beings. Accordingly, a patientto be treated with certain of the novel compounds of this invention isfirst diagnosed as a diabetic by conventional means (e.g., thepersistence of elevated serum glucose levels), and a treatment regimenwith certain compounds of this invention established so that theelevation in a patient's serum glucose level is either significantlyreduced or eliminated. The precise therapeutic endpoint of treatment(i.e., elimination or merely reduction in hyperglycemia) is readilydetermined by the attending physician based upon the clinicalpresentation and concomitantly employed treatment. For example, certainof the novel compounds of this invention may be employed tosignificantly reduce hyperglycemia in a patient, with acarbohydrate-restricted diet providing the further measure of control.

While the novel compounds of this aspect of the invention may beadministered by any convenient route) e.g., orally, subcutaneously,intravenously, intramuscularly, topically, or rectally), these compoundsare most significantly and usefully employed as oral hypoglycemicagents, particularly in solid dosage form, e.g., capsules and tablets.Alternatively, liquid oral dosage forms (e.g., syrups and elixirs) arealternatively employed. The solid, oral pharmaceutical compositions inaccordance with the present invention are all prepared by methods knownin the art, e.g., methods for preparing other oral antidabeticcompositions. Since an individual patient response to treatment withcompounds in accordance with the present invention may vary, effectivedosages of the compounds of the instant invention will vary from patientto patient. Ordinarily, an oral dosage of from 0.1 to 10 mg/kg of acompound in accordance with the instant invention will be adequate tosignificantly reduce hyperglycemia in a patient being treated. Repeateddosages, e.g., every 4-12 hr, may be required during the day to maintainthe antihyperglycemic effect. Accordingly, dosages in accordance withthe present invention may range from as low as about 0.1 mg/kg/dose toas high as about 10 mg/kg/dose, depending upon the patient, frequency oftreatment, and observed response. In accordance with well-recognizedmethods, an attending physician may at first prescribe a relativelysmall amount of the novel compound of this invention, with subsequentincreases in this dosage as necessary to achieve the desired level ofcontrol.

Some of the compounds of the present invention are also useful to treatand/or prevent obesity in mammals including human beings. For thispurpose, the novel compounds of this invention are formulated andadministered as described above for hyperglycemia.

DETAILED DESCRIPTION

The definitions which follow apply throughout the specification unlessspecifically noted otherwise.

All temperatures are in degrees Centigrade.

Halogen refers to chlorine, bromine, fluorine or iodine.

Hal refers to chlorine, bromine, or fluorine.

IR refers to infrared spectroscopy.

NMR refers to nuclear (proton) magnetic resonance spectroscopy, chemicalshifts are reported in ppm (δ) downfield from TMS.

TMS refers to tetramethylsilane.

MS refers to mass spectrometry.

Ts refers to p-toluenesulfonyl.

Ms refers to methanesulfonyl.

Brine refers to saturated aqueous sodium chloride.

R¹ -R¹⁶ and X¹ -X⁹ are as defined in the first occurrence of each hereinunless indicated otherwise. For example R₄ as used in claim 2 has thesame definition as R₄ used in claim 1.

The charts are meant to be schematic and the variables used therein areused generically. For example R may be alkyl, X⁻ may be halogen, etc.

When the term "Cn-Cp alkyl", for example, is used, it means and includesisomers thereof where such exist.

A bond indicated as "˜" includes both the α and β configurations.

Acyl includes aroyl, phenacyl, substituted phenacyl, COCH₃, COC₂ H₅, andCOC₃ H₇.

Method For Preparing Compounds of Formula III and Related Compounds

Referring now to Chart A:

The formamide A-2 may be prepared from the Leuckart reaction of formulaA-1, e.g., estrone methyl ether with 3-dimethylaminopropylamine informic acid. To purify the crude reaction product, chromatography (e.g.,silica gel, 5% of a 10% NH₄ OH--CH₃ OH solution in CH₂ Cl₂, see sectionbelow on reactions of amino-side chains upon prolonged contact with CH₂Cl₂) is helpful.

When the synthesis of formamide A-2 is modified to use, for example, aratio of estrone methyl ether:amine:formic acid of 1:5:5, the reactionyields the un-formylated 17-NHR formula A-3.

To obtain the N-methyl group of formula A-4, amide A-2 is reduced withlithium aluminum hydride (LiAlH₄) in dioxane according to knownprocedures. The crude product obtained from this reduction is of highquality and may be used directly for most subsequent transformations,including salt formation and reduction.

Reaction of Dimethylaminopropyl Side Chains with Methylene Chloride

Referring now to Chart A:

When solutions of, for example, steroids containing tertiary amines inmethylene chloride are allowed to stand at 25° for several days, a muchmore polar product, the methylene chloride adduct, A-7 accumulates. Theproduct is isolated by trituration with 90% ethyl acetate/hexane.

Treatment of the methylene chloride adduct A-7 with excess sodiumethoxide in refluxing ethanol affords the same N-allyl product, A-8, asthat obtained, infra, from mild pyrolysis of the N-oxide A-5, as well asthe N-demethylated analog.

Quaternary Amine Salts and N-Oxides

Referring now to Chart A:

Reaction of formula A-4 with, for example, excess methyl iodide inmethanol gives an insoluble solid compound which has a mono quaternaryammonium iodide structure A-7.

Alkylation of A-4 with benzyl chloride and with ethyl chloroacetategives quaternary salts. In all of these cases, quaternization occurs atthe terminal nitrogen of the side chain in preference to the 17-aminonitrogen.

Synthesis of the mono- and bis-N-oxides of formula A-4, is set forth inChart A, Step A-4. Compound A-4 is oxidized, for example, with hydrogenperoxide in methanol (A. C. Cope and Ciganek, Org. Synth.Coll., 4, p.612 (1963)), a reaction which produces selectively either the mono-,A-5, or bis-N-oxide, A-6, depending on the amount of hydrogen peroxideemployed and the reaction time.

A-Ring Modifications of Compound A-10

Referring now to Chart A:

For A-ring modifications of the steroidal moiety, Birch reduction ofmethoxy A-9 gives the more highly saturated compound A-10. To reduce thepresence of small amounts of any unreduced starting compound in theproduct, several recrystallizations from etheracetonitrile may beemployed.

To produce compounds of the present invention with the methoxysubstituent removed from the A-ring of the steroidal moeity, forexample, 17-ketoestrane is subjected to the Leuckart reaction-LiAlH₄reduction sequence, (Chart A, steps 1 and 2, supra) to produce theformamide and the diamine.

Referring now to Chart D:

Acetate and butyrate derivatives (D-4) of phenol D-2 can also beprepared, and the phenol D-2 also can be brominated withN-bromoacetamide to produce the 4-bromo analog D-5.

We have also prepared a series of analogs in which the diaminoside-chain is modified in various ways. This series of compounds has thearomatic A-ring rather than the dihydro aromatic A-ring to avoid theBirch reduction step. The aromatic A-ring analogs further avoid thechemical instability inherent in the enol-ether of the dihydro aromaticcompounds.

In preparing the side-chain modifications, two synthetic routes wereused. The first of these is the Leuckart reaction of, for example,estrone methyl ether with the desired amine in formic acid as outlinedin Chart A, supra.

The second synthetic reaction is the reductive amination of, forexample, estrone methyl ether with the desired amine and sodiumcyanoborohydride (Chart B), R. F. Borch et al., J. Am. Chem. Soc., 93,p. 2897 (1971). For this series of analogs we used only primary aminesin the reaction, consequently the products are the secondary aminesrepresented by formula B-2.

We have also prepared various analogs in which the major changes are inthe alteration of the steroidal (or hydrocarbon) nucleus of themolecule. Various groups such as cyclopentadecyl, cyclododecyl, decalyl,adamantyl, bicyclohexyl, and pyrenyl have been used to substitute forthe estrogen steroidal skeleton.

We began by selecting various readily availabe molecules that werecomprised primarily of a large hydrocarbon portion and a functionalgroup that could easily be converted into a diamino sidechain. Severalmacrocyclic ketones are commercially available and provide a convenientpoint of departure.

Referring now to Chart A:

The first analog of this type was obtained from the Leuckart reactionbetween cyclopentadecanone and 3-dimethylaminopropylamine in formic acidwhich gave the formamide (A-2). Also, the secondary amine (A-3) whichhas not undergone formylation during the reaction may be isolated.During chromatographic purification of the secondary amine on silica gelusing ethyl acetate-methanol-triethylamine, the compound is obtainedfrom the column as the acetate salt. Reduction of the formamide A-2 withlithium aluminum hydride gives the N-methyl analog A-4 which isconverted to a crystalline disuccinate salt.

We have also prepared a similar analog from cyclododecane using thereductive amination procedure with NaB(CN)H₃ (Chart B, Step 1) insteadof the Leuckart reaction to attach the diamino side-chain. As in thecase of the secondary amine above, this secondary amine also is obtainedas a crystalline acetate salt following chromatography.

Several other side-chain modifications have been incorporated with thesemacrocyclic hydrocarbons. The bisdimethylaminopropyl amine side chain,has been attached to cyclododecane and a tetraamino side chain has beenattached to cyclopentadecane.

Bicyclohexyl Hydrocarbon Analogs

The preceding macrocyclic analogs are the least rigid cyclic systemsavailable. Using other available ketones, we have prepared analogs withincreasing degrees of structural rigidity. Reductive amination of2-cyclohexylcyclohexanone and 4-cyclohexylcyclohexanone gives in eachcase the isomeric cis and trans compounds. The isomers are separated bychromatography.

Three isomeric pairs of analogs, from reaction with3-dimethylaminopropylamine, 1,4-diaminobutane, and 3-aminomethylpyridinehave been prepared from 4-cyclohexylcyclohexanone. Another pair ofanalogs closely related to the above was obtained from reaction of4-phenylcyclohexanone with 3-dimethylaminopropylamine.

Rigid Hydrocarbon Analogs

trans-1-Decalone and adamantanone are two readily available ketoneshaving ridid hydrocarbon skeletons. Both have been converted to3-dialkylaminopropylamine derivatives. The former when aminated with3-diethylaminopropylamine gives a single compound. Reaction ofadamantanone with 3-dimethylaminopropylamine gave a product which wasconverted to the crystalline disuccinate salt.

Other Hydrocarbon Analogs

One of the first modified analogs we prepared used a steroid,3-cholestanone, as the hydrocarbon nucleus. Leuckart reaction conditionswith 3-dimethylaminopropylamine produces a mixture of formamides whichwere reduced to the methylamines. No separation of isomers could bedetected with various chromatographic systems, so the mixture wasconverted to the solid disuccinate salt.

Pyrene-1-carboxaldehyde was converted to the diamine derivative as anexample of a polycyclic aromatic hydrocarbon analog.

Many new nonsteroidal anti-inflammatory agents have been developed inrecent years and these frequently contain a bicyclic hydrocarbon nucleusattached to a propionic acid. These are converted to diamines via theacid chlorides and amides. We converted flurbiprofen to the diamine bypreparation of the acid chloride followed by reaction with3-dimethylaminopropylamine to obtain the amide. Lithium aluminum hydridereduction of the amide produced the diamine. Another analog was preparedby similar treatment of the3-ethyl-4-(p-methoxyphenyl)-2-methyl-3-cyclohexene-1 carboxylic acid.

The first analog prepared containing a pyridine ring as a part of thediamino side-chain was obtained from the Leuckart reaction of estronemethyl ether with 3-aminomethylpyridine (Chart A, Step 1). This reactiongives the expected formamide A-2 together with the secondary amine(A-3), supra.

Compounds such as these can also be prepared by the reductive aminationof estrone methyl ether with the desired amine and sodiumcyanoborohydride (Chart B) which is a simpler route to the compoundsthan the Leuckart reaction. Good yields are observed for compoundsproduced this way and only in some cases is a chromatography stepemployed. Analogs having a phenyl or a substituted phenyl ring in placeof the pyridine can also be prepared in this way.

When the Leuckart reaction between estrone methyl ether and2-(2-aminoethyl)pyridine is attempted, the reaction product isN-formyl-3-methoxyestra-1,3,5(10)-trien-17β-amine rather than theexpected formamide. The 2-(2-aminoethyl)pyridine side chain can beattached to estrone methyl ether by reductive amination.

The 3-aminomethylpyridine group can be added to cyclopentadecanone togive an analog and to 4-cyclohexylcyclohexanone to give two isomericanalogs.

Variation in Side Chain Length

A homologous series of α,ω-diamines was synthesized having a variablenumber of carbons between the nitrogen atoms. Most of these analogs wereprepared via reductive amination (R. F. Borch et al., J. Am. Chem. Soc.,93, p. 2897 (1971) of the ketone of formula I (purchased from Searle)with the appropriate diamines (in most cases, commercially available).

Also synthesized were a series of compounds in which the terminalnitrogen atom of the steroidal diamines is derivatized. Conversion ofN-(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,6-hexanediamine to a seriesof derivatives is accomplished in good yields using standards methods.

Reductive amination of the estrone 3-methyl ether with methylamine wasdone on a reasonably large scale (>10 g) and provided both the 17α- and17β-isomers in a ratio of 1:12. The 17β-isomer in particular is a keyintermediate en route to certain other diamines, via amide formationusing the C-17 amine, followed by LiAlH₄ reduction.

Several tri- and tetra-amino steroids were prepared. In each case, therequired polyamine fragment was commercially available. This seriesoffered variety both in terms of side chain polarity and metal chelatingability.

The synthesis of analogs with the diamine moiety attached at C-20 of aprogesterone side chain was also done. The conversion of estrone3-methyl ether to 17-deoxo-17-acetyl derivative was accomplished by theprocedure of Bull and Tuinman Tetrahedron, 31, p. 2151 (1975). The C-17ketone was transformed into the corresponding 17-cyano steroid viacondensation with tosylmethyl isocyanate. The nitrile was then treatedwith methyllithium to afford 17β-acetyl intermediate along withapproximately 20% of the 17α-isomer, readily separablechromatographically. Subjection of the 17β-intermediate to theLeuckart/LiAlH₄ sequence led to the recovery of two isomeric diamines.NMR evidence suggested that some loss in stereochemical purity hadoccurred at C-17, so that four isomers were possilbe. Reductiveamination of the 17β-intermediate with N,N-1,3-diaminopropane (undermuch milder conditions) gave only the two expected C-20 epimers.Treatment of the 17β-intermediate under the standard Mannich conditionsafforded a mixture of the desired aminoketone and elimination product.

A variety of other steroid-based analogs were also synthesized. Theornithine ester was prepared via water-soluble-carbodiimide mediatedesterification of bis (BOC) ornithine with steroidal 17β-alcohol,followed by deprotection of the amines with trifluoracetic acid.

Alkylation of the same alcohol with dimethyl trimethylene ammonium saltgave the amino ether. The recovery of the product in the form of itsmethanesulfonate salt results from the use of sodium bicarbonate ratherthan sodium hydroxide in the work-up.

N-[3-(Dimethylamino)propyl]-3-methoxyestra-1,3,5(10)-trien-17α-amine wassynthesized by displacement of a 17β-mesylate withN,N-dimethyl-1,3-diaminopropane (V. V. Runade et al., J. Med. Chem., 14,p. 38 (1971)). By far the predominant product in this reaction was theΔ¹⁶ -elimination product.

A steroidal diamine with an 11β-hydroxyl and an unfunctionalized, fullysaturated A ring, was made via the standard Leuckart reaction.

Inhibition of Rat Neutrophil Aggregation

1. Method for Thioglycolate Broth Preparation

Weigh sufficient thioglycolate medium, USP grade, to prepare a 5% w/vsolution in sterile water. Heat the solution for 10 minutes on a boilingwater bath. Remove and allow the solution to cool to 20°-25°. Inject10±0.5 ml intraperitoneally into Sprague-Dawley rats as described below.

2. Method for Rat Peritoneal Leukocyte Collection

Six (6) Sprague-Dawley, pathogen free, female rats (230-270 grams) areinjected intraperitoneally with 10.0±0.5 ml thioglycolate broth, 5% w/v16-18 hours prior to sacrifice. After sacrifice by cervical dislocation,leukocytes accumulated in the peritoneal cavity are collected byinjecting 30 ml of sterile 0.9% w/v sodium chloride intraperitoneallyand vigorously massaging the abdomen to assure uniform dispersion of thecells within the carcass. Use a pasteur pipet to remove approximately 20ml of fluid with suspended cells from a small incision through theabodominal wall. Collect the cell suspension in plastic culture tubes.

3. Washing and Resuspension of Cells for Aggregation

Centrifuge the cell suspensions isolated above for 10 minutes at 1000rpm (Sorvall RC-3, HG-4L rotor, 25° C.). Discard the supernatant.Resuspend the cells evenly in 0.9% NaCl to original volume, centrifuge asecond time for 10 minutes at 1000 rpm. Discard the supernatant.Resuspend the cells evenly in Hanks buffer.

4. Determination of Leukocyte Concentration

Transfer 10 μl of leukocyte suspension into a plastic cell counting cup.Add 15.0 ml of ISOTON˜ diluent for cell counting. Determine the cellcount with a Model ZBI Coulter Counter or equivalent.

5. Neutrophil Aggregation

A. Add 0.5 ml of rat leukocyte (neutrophil) suspension to each channelof a Payton dual channel aggregometer. Cuvettes, 45 mm×4 mm i.d. areused. Cell suspensions at 37° C. are stirred (400 rpm).

B. Add 5 μl test compound (0.01M in absolute ethanol) to cell andevaporate to dryness under nitrogen. Add 0.5 ml cell suspension (37° C.,400 rpm). Incubate for 2 minutes, then add 1 μl of the agonist, 10⁻⁴ MFMLP.

C. Record the aggregation trace (% transmitted light) on apotentiometric recorder.

Inhibition of Hog Pancreas PLA₂

1. Enzymes

Both soybean lipoxidase and hog pancreas phospholipase A₂ are obtainedcommercially from Sigma. The soybean lipoxidase is disolved at aconcentration of 5 mg/ml in 0.033M ammediol-HCl buffer pH 8.5 with1×10⁻⁴ M Ca⁺⁺. The hog pancreas enzyme is added at the rate ofapproximately 350 units per ml of final mixture. Thus, 0.025 ml isequivalent to 9 units of phospholipase and 0.125 mg of lipoxidase.

2. Substrate

The substrate is phosphatidyl choline. The material has a fatty acidcomposition upon saponification, of 2% of 16:0, 1% of 18:0, 3% of 18:1,18% of 18:2, and 12% f 18:3 fatty acids with the largest fraction beinglinoleic acid. The estimated molecular weight is 780.

78 mg of this substrate is put in a 10 ml volumetric flask containing100 mg of deoxycholic acid. A "pill" magnetic stirrer is added alongwith 7-8 ml of water, and the whole stirred rapidly until all thelecithin is dissolved. The "pill" is then removed and the flask contentsare made up of 10 ml with water.

3. Procedure

To three oxygraph cells equipped with magnetic stirrers is added 2.5 mlof 0.033M ammediol-HCl buffer pH 8.5 containing 1×10⁻⁴ M Ca⁺⁺. This isfollowed by 0.1 ml of the inhibitors made up at an initial concentrationof 0.01M in methanol. Where controls are run, 0.1 ml of methanol isadded to each cell. The cells are then put in the oxygraph apparatus andthe contents are stirred briefly. 0.025 ml of the enzyme mixture is thenadded and the electrodes are inserted in each cell, care being taken toexclude all air bubbles. With the stirrer and water bath pump on, thecontents of each cell are stirred for 2.5 minutes at 37.5° C. Thereaction is initiated by adding to the cells 0.05 ml of 0.01M lecithinsubstrate. The reaction is monitored by continuous measurement of ratesof oxygen depletion from the medium as a consequence of unsaturatedfatty acids (linoleic acid) being released from esterified form by thephospholipase. These fatty acids immediately become substrates for thesoybean lipoxidase which forms the 15-hydroxy acids, with consequentoxygen utilization.

The inital rates of oxygen consumption are recorded using aSargeant-Welch Recorder set at 5 mV full scale. The "air" setting andmedium chart speed are used. The slopes of oxygen consumption are thendetermined in triplicate, and these are compared with the methanolcontrols to determine the degree of inhibition. If complete inhibitionis seen at the first concentration, appropriate dilutions are made tobring the inhibition percentages down to a least 3 concentrations ofinhibitor where partial inhibition is observed. The I₅₀ can then becalculated for that particular inhibibor, using linear regressionslopes. All compounds for which I₅₀ value is shown are tested forinhibitory activity on the soybean lipoxidase. None inhibit at the testconcentration.

Testing For Blood Glucose Lowering In The KKA^(y) Mouse

1. General

All KKA^(y) mice used for screening are produced and selected by methodspreviously outlined, T. Fujita et al., Diabetes, 32, 804-810 (1983).Groups of 6 animals each are employed.

2. Screening Procedure

Pre-treatment nonfasting blood glucose (NFBG) samples are measured 5days prior to the start of a screening run by previously describedmethodologies. These blood sugar values are used to place animals intogroups with equal mean blood glucose concentrations and to eliminate anymice with a NFBG value <250 mg/dl. On day 0, compounds chosen to be runare incorporated into ground mouse chow (Purina 5015). Compounds areincluded at a rate of 1 mg/gram of chow. Generally, 300 g of drugscontaining diet is prepared for each group. Mice receiving ground chowonly are the negative control. Each screening run also uses ciglitazone(T. Fujita, et al., supra) as a positive control (0.5 to 1.0 mg/gramchow).

Initial body and food weights are taken on day 1. Food is placed in acrock which contains an adequate amount to last for the length of thestudy. In order to acclimate the mice from pelleted mouse chow to groundmouse chow, they are fed the ground chow for 9 days prior to use in thescreen. On day 4 of treatment, a NFBG sample is again measured, as wellas food and body weights. Food consumption measurements are used todetermine an average mg/kg dose the mice received over the testingperiod, and to evaluate the compound's effect on food consumption.

3. Acceptance of a Screening Run and Determination of Activity

Acceptance and activity are determined by the following criteria:

A. Negative Control

This group must not show a significant change (p<0.05) from pre- topost-treatment. If there is a significant decrease in blood sugar itmeans the run is not valid.

B. Positive Control

This group must show a significant depression in blood sugar mean levelsfrom pre- to post-treatment. A lack of activity in this group would alsoinvalidate the run.

C. Negative Control vs. Positive Control

This contrast must be signifcant. It is a further assurance that bothcontrol groups performed as expected.

D. Compound

A compound's activity is based on several criteria:

(1) A significant decrease in blood sugar mean levels from pre- topost-treatment.

(2) Negative control vs. compound: This contrast allows one to determineif these groups are dissimilar, which is required for the compound to beconsidered active.

One skilled in the art may determine the specific utility of eachcompound of the present invention by assaying them by the precedingmethods and for angiogenic and angiostatic activity, infra. Compoundsthat have been found to have a phospholipase A₂ inhibitory orantidiabetic effect as determined by at least one of the above assaysare indicated in the Examples and Preparations which follow by thenotation "PLA2" and/or "diabetes" respectively. If the compound has beentested as inactive in all tests for one of the respective activities,that is indicated by "PLA2-" or "Diabetes-" respectively. Some of thecompounds were assayed for PLA2 activity by more than one method. If anymethod showed activity, that compound is listed as having PLA2 activity.

The preferred compounds for phospholipase A2 inhibition include1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dioneN-[3-(3-aminopropoxy)estra-1,3,5(10)-trien-17-yl]N,N',N'-trimethyl-1,3-propanediamineandN-[3'-(dimethylamino)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amine.

The preferred compounds for antidiabetic activity include3-methoxy-17β-[((3-trifluoromethyl)phenylmethyl)-amino]-estra-1,3,5(10)-triene,N-[3'-(dimethylamino)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amineand 3-methoxy-17β-[(4-chlorophenylmethyl)amino]-estra-1,3,5(10)-triene.

Preparation 1N-[3-(Dimethylamino)propyl]-[N]-formyl-3-methoxyestra-1,3,5(10)-trien-17.beta.-amine

Formic acid (95-97%, 100 ml) is added dropwise to a cooled and stirredmixture of estrone methyl ether (71.0 g) and 3-dimethylaminopropylamine(158 ml). After completing the addition, the mixture is warmed in an oilbath. At 160°-170° C. bath temperature, considerable gas evolution isobserved and care must be taken so that the reaction does not froth outof the flask. When the strongest bubbling subsides, the bath temperatureis increased to about 175° C. and heating continued for 18 hours. Thereaction mixture is poured onto ice water (500 ml), 50% aq. NaOH (75 ml)is added, mixed well, and stirred with CH₂ Cl₂ (500 ml). The layers areseparated and the aqueous phase is extracted with 250 ml CH₂ Cl₂. Thecombined organic extracts are washed with brine, dried (Na₂ SO₄),filtered, and concentrated to give an oil. A portion of the crudeproduct (44.4 g) is chromatographed on silica gel (2 kg) packed andeluted with 5% of 10% NH₄ OH--CH₃ OH in CH₂ Cl₂. Fractions of 375 mlvolume are collected and the desired product is eluted in fractions39-82. These are pooled (25.9 g) and crystallized.

Additional crude product from the above reaction and from a secondsimilar reaction is chromatographed in two portions. From these columns,the title compound is obtained. each lot is dissolved in hexane (-500ml) and acetone (50 ml), filtered, and reduced in volume. Cooling givescrystalline title compound in two lots, mp 102°-104° C., and mp100°-103° C. (Diabetes)

Preparation 2N-[3-(Dimethylamino)propyl]-3-methoxy-N-methylestra-1,3,5(10)-trien-17.beta.-amine

Under a N₂ atmosphere, a mixture of lithium aluminum hydride (7.0 g) indioxane (300 ml) is stirred at 20°-25°. To this is added a solution ofthe title compound from Preparation 1 (30.5 g) in dioxane over a periodof 10 minutes. The resulting mixture is warmed in an oil bath to 100° C.over a perid of 90 minutes. In reductions of amides such as this, asudden evolution of gas in the temperature range of 80°-100° C. isfrequently observed with much frothing of the mixture. Care must betaken to allow for this expansion or material may be lost from thereaction vessel. After determining by TLC that the reduction iscomplete, the reaction is cooled and quenched by the careful addition ofaqueous sodium sulfate (75 ml) and water (150 ml). After stirring 45min., excess solvent is removed under reduced pressure. Methylenechloride (750 ml) and sodium sulfate are added to the residue. Themixture is mixed thoroughly, the methylene chloride is separated, andthe solids washed twice more with additional methylene chloride. Thecombined CH₂ Cl₂ extracts are concentrated, giving the title compound asan oil. ¹ H NMR (CDCl₃) δ7.24, 6.72, 6.67, 3.77, 2.82, 2.23, 3.80. (PLA2and diabetes)

Preparation 3N-[3-(Diaminomethyl)propyl]-3-methoxy-N-methylestra-1,3,5(10)-trien-17.beta.-amineDisuccinate

A solution of succinic acid (6.13 g) in warm methanol (50 ml) is addedto a solution of the title compound from Preparation 2 (10.0 g) inmethanol (65 ml). The solution is mixed well and left at 20°-25°.Crystals form and are collected giving the title compound, mp 169°-172°C. (Diabetes)

Preparation 4N-[3'-(Dimethylamino)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amine

Liquid NH₃ (1775 ml) is introduced into a three-neck, 5 1 flask equippedwith an air-driven stirrer, a N₂ inlet, and a dry ice/acetone condenserwhile cooling the flask in an acetone/dry ice bath. The cooling bath isremoved, the solution is stirred, and ether (800 ml) is added to theammonia. Lithium wire (13 g) washed in toluene to remove oil, is cut inpieces and added slowly to the stirred solution. The lithium dissolvesquite rapidly and produces a deep blue solution with a bronze-color castat the glass wall surface. The title compound from Preparation 2 (34.3g) is dissolved in ether (500 ml) and added via a dropping funnel,inserted between flask and N₂ inlet, over a period of 40 minutes. Theresulting solution is stirred at the reflux temperature of the ammoniafor 30 minutes and then absolute ethanol (170 ml) is added slowly untilthe solution turns a cloudly white color. Excess ammonia is removedunder a stream of N₂ by warming the flask on a steam bath. When theresidue consists primarily of ether and white solids, it is transferredto a 2 1 k separatory funnel with the aid of additional ether.Brine-water (1:1) is added cautiously (much heat evolves) until twoclear layers are seen. The aqueous layer is separated and the etherlayers is washed twice with brine, dried (MgSO₄) and degassed withargon. Following drying, the mixture is filtered, the ether filtrate isconcentrated, and the crude crystalline residue is recrystallized fromether-acetonitrile giving the title compound, mp 78°-80° C. An NMRspecturm reveals a small amount of starting material to be present inthe product. A second recrystallization from ether --CH₃ CN gives thetitle compound, mp 80°-81.5° C. and a third recrystallization(ether--CH₃ CN) gives the title compound, mp 81°-82° C. (PLA2 anddiabetes)

Preparation 5N-[3'-(Dimethylamino)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amineDisuccinate

Succinic acid (1.23 g) is dissolved in a methanol (18 ml) solution ofthe title compound from Preparation 4 (2.00 g) by warming on a steambath. Upon cooling, crystals form and are collected. Recrystallizationfrom methanol-ether gives the title compound as crystals, mp 162°-165°C. (PLA2)

Preparation 6 Ornithine methyl ester dihydrochloride

The procedure of Golankiewicz and Wiewiorowski, Acta BiochimicaPolonica, 10, p. 443 (1963), is utilized. A 50 ml round-bottomed,3-necked flask, equipped with a magnetic stirrer and a gas dispersiontube, is flame dried and then cooled in an atmosphere of nitrogen. Theflask is charged with 2 g of ornithine hydrochloride and 25 ml ofmethanol. The solution is then saturated with hydrogen chloride gas(anhydrous) and concentrated in vacuo (50° C., 1.0 mm, 30 min). Theprocedure is repeated as before with the exception of a longer timeconcentrating in vacuo (5 h).

The resulting white hydroscopic solid is kept in a nitrogen atmosphereand yields the title compound; NMR (CDCl₃, TMS) δ4.3-4.1, 3.9, 3.15-2.85and 2.2 ppm-1.7 ppm.

Preparation 7 3-Methoxy-17(α and β)-cyano-1,3,5(10)-estratriene

The procedure used is taken from J. Bull and A. Tuinman, Tetrahedron,31, p. 2151 (1975). A flame dried 3000 ml 3-neck round-bottomed flask,equipped with a magnetic stir bar, two constant pressure additionfunnels, and a nitrogen inlet tube, is charged with estrone methyl ether(9.0 g) and 800 ml of glyme. Commerically available potassiumtert-butoxide (73.0 g, Aldrich) is dissolved in 600 ml of tert-butanoland added rapidly to the starting material. After complete addition, thesolution is a clear yellow. Tosylmethyl isocyanide (12.7 g) in 200 ml ofglyme is added slowly (2 hours and 20 minutes) to the above solution at20°-25°. After an additional hour of stirring at 20°-25° TLC indicatesthe reaction is complete. The reaction is worked up by treating themixture with water (150 ml), brine (500 ml), and ethyl acetate (2000ml), and then separating the layers. The organic phase is washed withbrine and dried (MgSO₄ ), filtered, and concentrated to provide a solid.

The solid is chromatographed on 1,850 g of 70-230 mesh silica geleluting with 10% hexane/methylene chloride. An initial fraction of 4000ml is collected, followed by 50 ml fractions. After fraction 85, puremethylene chloride is used for further elution. By TLC, fractions 83-187are homogeneous and are combined and concentrated to give a solid (mp150°-185° C., wide range due to a mixture of the α and β isomers); NMR(CDCl₃, TMS) δ7.33-7.13, 6.83-6.6, 3.77, 3.0-1.1, 0.93 and 0.83.

Preparation 8 17β-Acetyl-3-methoxy-1,3,5(10)-estratriene

A flame dried 3-neck 2000 ml round-bottomed flask, equipped with amagnetic stir bar, nitrogen inlet tube, and 1000 ml constant pressureaddition funnel, is charged with the title compound from Preparation 7(11.66 g) and 600 ml of tetrahydrofuran. Addition of a methyl lithiumsolution (470 ml of a 1.6M solution as a complex with lithium bromide indiethyl ether, 0.752 mol) is carried out over 20 minutes with slightcooling of the reaction vessel using a cold water bath. The mixture isstirred an additional 1.5 hours at 20°-25° before workup. Analysis byTLC indicates little progress in the reaction over an additional onehour period, and increasing decomposition of products. The reaction isquenched with water (under nitrogen) and extracted with ethyl acetate.The organic phase is washed with 1M hydrochloric acid, saturated sodiumbicarbonate, and brine, dried (MgSO₄), filtered, and concentrated togive a solid.

The solid is chromatographed on 1300 g of 70-230 mesh silica gel elutingwith 20% toluene/methylene chloride. An initial fraction of 4000 ml iscollected followed by 40 ml fractions. After the starting material haseluted (fractions 83-112), the eluent is changed to pure methylenechloride. Fractions 148-182 are homogenous by TLC and correspond to theα-isomer. These fractions are combined and concentrated to provide asolid. The following 900 ml collected contains a mixture of the α and βisomers with only a trace of the αisomer. Solvent evaporation gives asolid.

The solid from the β-isomer is recrystallized several times from amethylene chloride-diethyl ether mixture to provide a solid, mp136°-137° C. (PLA2-)

Preparation 9 N,N'-Bis(t-butoxycarbonyl)-ornithine

A 100 ml round-bottomed, 3-necked flask, equipped with a magneticstirrer is purged with a stream of nitrogen. The flask is then chargedwith 4.6 g of ornithine hydrochloride dissolved in 40 ml of aqueousacetone (1:1).l To this solution is added 14.8 g oft-butyloxycarbonyl-oxyimino-2-phenylacetonitrile (BOC-ON) followed by12.4 g of triethylamine. The reaction mixture is stirred at 20°-25° for3 h.

The reaction mixture is concentrated in vacuo. The resulting residue ispartitioned between ethyl acetate and water (pH 3/acidified with 2Msodium bisulfate). The organic layer is separated, washed with brine,dried over magnesium sulfate and concentrated in vacuo.

The crude product is chromatographed on 600 g of CC-4 silica gel. Thecolumn is packed and eluted with 50% hexane/ethyl acetate (10 mlfractions).

Fractions 60-150 are combined based on their TLC homegeneity and affordthe title compound; NMR (CDCl₃, TMS) δ 10.5-10.2, 3.3-3.0 and 2.0 ppm-1.0 ppm.

Preparation 10 Dimethyltrimethyleneammonium methanesulfonate

A solution of 3-dimethylamino-1-propanol (10 ml, 8.72 g) in Burdick &Jackson methylene chloride (100 ml) is cooled to -30° C. and treatedwith triethylamine (14.44 ml) and then slowly over 10 minutes withmethanesulfonyl chloride (7.85 ml). The resulting white suspension isstirred for 40 minutes at -25° to -20° C., treated with ice, stirred for10 min, then partitioned between ethyl acetate (300 ml) and saturatedsodium bicarbonate (300 ml). The layers are separated and the aqueouslayer is extracted with ethyl acetate (2×500 ml). The combined organicsare washed with aqueous saturated sodium bicarbonate (300 ml), driedover magnesium sulfate, filtered and concentrated to an oil, whichgradually solidifies while being stored under high vacumm overnight; NMR(CDCl₃, TMS) δ 2.60-3.00, 3.43, 4.57.

Preparation 11N-[3-(Dimethylamino)propyl]-N-methyl-3-hydroxyestra-1,3,5(10)-trien-17.beta.-amine

A degassed solution of freshly distilled THF (60 ml) anddiphenylphosphine (2.09 ml) is cooled to 0° C. under nitrogen andtreated dropwise with n-butyllithium in hexane (7.08 ml). The resultingred solution is stirred for 5 minutes at 0° C. and for 30 minutes at20°-25° and is then treated at 20°-25° with a solution of the titlecompound from Preparation 3 (1.58 g) in freshly distilled THF (12 ml+3ml+3 ml). The resulting red solution is stirred at reflux for 5 hours,cooled to 0° C. and treated with diphenylphosphine (3.14 ml) followed by1.61M n-butyllithium in hexane (10.62 ml). The red solution is stirredat 0° C. for 5 minutes, at 20°-25° for 25 minutes and at reflux forabout 12 hours.

The reaction is cooled to 20°-25°, concentrated in vacuo andchromatographed on "flash chromatography" - 3"×101/2" of HPLC gradesilica gel in 1:15:85 ammonium hydroxide/methanol/methylene chloridewhile collecting 150 ml fractions. Fractions 5-11 are combined andconcentrated to give the title compound, mp 156°-157° C. (PLA2/diabetes)

Preparation 12 4-Cyclohexylcyclohexylidenephosphorohydrazidic Acid,Diethyl Ester

To a solution of 4-cyclohexylcyclohexanone (27.04 g) and acetic acid (3ml) in CH₂ Cl₂ (400 ml) was added a solution of diethylphosphorohydrazidate (28.91 g) in CH₂ Cl₂ (100 ml). The reaction wasstirred at room temperature for 27 hours and then was concentrated underreduced pressure. Toluene was added (2X) and removed under reducedpressure in order to aid in the removal of acetic acid bycodistillation. The residual yellow liquid was the product and was usedwithout further purification. TLC Rf=0.52 (20% acetone in CH₂ Cl₂,silica gel).

Preparation 13 1-(4'-Cyclohexyl)cyclohexylidenenhydrazone-4-(2",2"-dimethyltrimethylene)ketal-1,4-cyclohexanedioine

In a nitrogen atmosphere, solid sodium hydride (3.6 g) was added inportions over a 15 minute period to a solution of4-cyclohexylcyclohexylidenephosphorohydrazidic acid, diethyl ester (50g)in ether (500 ml). The mixture was stirred for 40 minutes and then asolution of 1,4-cyclohexanedione, mono-2,2-dimethyltrimethylene ketal(33.66 g, 0.195 mol) in ether (500 ml) was added dropwise over a periodof 20 minutes. After stirring at room temperature for 3 hours, TLCindicated that the reaction was incomplete. Another 0.15 mol of sodiumhydride was added and after stirring an additional 1.5 hour, thereaction was complete and was carefully quenched with water. The etherlayer was separated and the aqueous phase was extracted again withether. The combined extracts were washed with water, dried (Na₂ SO₄),filtered, and concentrated. The residue was a white, solid which wasused in the next Preparation without purification. TLC of the solidshowed an Rf=0.38 (40% ethyl acetate in hexane, silica gel).

Preparation 1411-Cyclohexyl-14,15-diaza-7-thiadispiro(5.1.5.2)-pentadecan-4-one(2',2'-Dimethyltrimethylene)ketal

The reaction flask was equipped with a balloon as an indicator of apositive H₂ S gas pressure. The flask was charged with a suspension of1-(4'-cyclohexyl)cyclohexylidenenhydrazone-4-(2",2"-dimethyltrimethylene)ketal-1,4-cyclohexanedione(0.15 mol) in 1:1 acetone-benzene (200 ml). Hydrogen sulfide gas wasadded from a lecture bottle so that the balloon attached to the flaskwas inflated. The suspension was stirred vigorously and H₂ S was addedperiodically in order to keep the balloon inflated. The mixture wasstirred at room temperature for 20 hours after which TLC indicated thatthe starting material had been completely converted to a new, more polarproduct. The mixture was filtered to remove solids, which were washedthoroughly with 1:1 actone-benzene. The filtrate was concentrated underreduced pressure giving the title compound as a semi-solid residue whichwas used without purification. TLC Rf=0.26 (40% ethyl acetate-hexane,silica gel).

Preparation 1511-Cyclohexyl-14,15-diaza-7-thiadispiro(5.1.5.2)-pentadec-14-en-4-one(2',2'-Dimethyltrimethylene)ketal

Powdered calcium carbonate (102 g) was suspended in hexane (1.5l), leadtetra-acetate (99.8 g) was added, and the mixture was stirred at 0° C.for 30 minutes. The hydrazine 11-cyclohexyl-14,15-diaza-7-thiadispiro(5.1.5.2) pentadecan-4-one (2',2'-dimethyltrimethylene)ketal (0.15 mol)in hexane (2.5 1) (not completely soluble so the insoluble solids wereadded as a suspension) was added over a period of one hour at 0° C. Thereaction mixture was stirred and allowed to warm to 20° C. over atwo-hour period. A TLC at this time showed the reaction to be complete.Saturated aqueous sodium bicarbonate was added, the mixture wasfiltered, and the filtrate was washed with saturated NaHCO₃ and withwater and then dried over Na₂ SO₄. The dry solution was filtered andconcentrated under reduced pressure, giving 45.5 g of solid. This solidwas chromatographed over silica gel (1.9 kg, 5% ethyl acetate-hexane)and the desired product was eluted after increasing the solvent polarityto 40% ethyl acetate-hexane. A sample was recrystallized twice frompentane, giving white crystals: IR 1586, 1448, 1446, 1439, 1367, 1363,1290, 1258, 1152, 1136, 1124, 1110, 1058, 1045, 1018, 980, 972, 967,955, 910, 904 cm⁻¹.

Preparation 16 11-Cyclolhexyl-7-thiadispiro(5.1.5.2)pentadecan-4-one(2',2'-Dimethyltrimethylene)ketal

The dry, solid thiadiazine, 11-cyclohexyl-14,15-diaza-7-thiadispiro(5.1.5.2) pentadec-14-en-4-one (2',2'-dimethyltrimethylene)ketal (10.72g, 0.0026 mol), was placed in a round bottomed flask under a N₂atmosphere. The solid was heated at 140° C. for 45 minutes. Aftercooling to room temperature, the solid was recrystallized from ethylacetate, giving the episulfide11-cyclohexyl-7-thiadispiro(-5.1.5.2)pentadecan-4-one(2',2'-dimethyltrimethylene)ketal, mp 212°-214° C. Recrystallization ofthe mother liquors followed by chromatography gave olefin4-(4'-cyclohexylcyclohexylidene)cyclohexane(2",2"-dimethyltrimethylene)ketal, mp 176°-177° C. IR (nujol) 1442,1366, 1362, 1354, 1313, 1286, 1280, 1247, 1240, 1233, 1218, 1140, 1129,1115, 1095, 1054, 1040, 1017, 962, 956, 910, 899, 891, 738 cm⁻¹.

Preparation 17 4-(4'-Cyclohexylcyclohexylidene)cyclohexane(2",2"-Dimethyltrimethylene)ketal

Method A. Solid thiadiazine,11-cyclohexyl-14,15-diaza-7-thiadispiro(5.1.5.2)pentadec-14-en-4-one(2',2'-dimethyltrimethylene)ketal (0.501 g) and triphenylphosphine(0.381 g) were placed in a round bottomed flask under a N₂ atomsophereand heated at 100° C. for two hours. The mixture was stirredoccasionally with a glass rod. The temperature was increased to 120° C.for an additional two hours. TLC at this time indicated completereaction. The cooled reaction mixture was chromatographed (one MerckLobar size B silica gel column, 5% ethy acetate-hexane, 20 ml fractions)and the title compound was eluted in fractions 8-13. Recrystallizationfrom hexane gave the title compound, mp 176.5°-177° C.

Method B. Raney nickel active catalyst (39 g, Aldrich slurry in water)was added in portions to a solution of episulfide11-cyclohexyl-7-thiadispiro(5.1.5.2)pentadecan-4-one(2',2'-dimethyltrimethylene)ketal (4.83 g) in 1:3 tetrahydrofuran-ethylacetate under an argon atmosphere. TLC showed the reaction to becomplete within 20 minutes. The reaction mixtures was filtered throughCelite and the filter cake was washed well with ethyl acetate. Thefiltrate was concentrated under reduced pressure giving the titlecompound.

Preparation 18 4-(4'-Cyclohexylcyclohexylidene)cyclohexanone Dimethylketal

A catalytic amount of p-toluenesulfonic acid (26 mg) was added to asolution of 4-(4'-cyclohexylcyclohexylidene)cyclohexane(2",2"-dimethyltrimethylene)ketal (5.56 g) in 1:1 methylenechloridemethanol (400 ml). TLC after three hours indicated the reactionto be about 50% complete. Additional methanol (200 ml) was added and thereaction checked by TLC after another two hours. About 10% of startingmaterial remained. The reaction was worked up at this time by theaddition of more CH₂ Cl₂ followed by saturated aqueous NaHCO₃ solution.The mixture was shaken and the layers were separated. The aqueous layerwas extracted two more times with CH₂ Cl₂, the pooled CH₂ Cl₂ extractswere washed with brine, dried (Na₂ SO₄), filtered, and concentrated to asolid. This dimethyl ketal was used without further purification.

Preparation 19 4-(4'-Cyclohexylcyclohexylidene)cyclohexanone

A solution of 4-(4'-cyclohexylcyclohexylidene)cyclohexanone dimethylketal (0.016 mol) and 1.0 N HCl (100 ml) in tetrahydrofuran (125 ml) wasleft at room temperature for 2.5 hours at which time TLC indicatedhydrolysis to be complete. Water and brine were added to the reactionsolution and the mixture was extracted with ether (4X). The pooled etherextracts were washed with brine, dried (Na₂ SO₄), filtered, andconcentrated to give 4.49 g of white solid. This solid waschromatographed (790 g of 40-63 μm silica gel, 4.5 1 of 5% ethylacetate-hexane followed by 10% ethyl acetate-hexane) with4-(4'-cyclohexylcyclohexylidene)cyclohexanone dimethyl ketal (0.56 g,10%) eluted first followed by the title compound. Recrystallization ofthe title compound from hexane gave colorless crystals, mp 81°-81.5° C.

EXAMPLE 1N-[3-(Dimethylamino)propyl]-3-methoxyestra-1,3,5(10)-trien-17β-amineDihydrochloride, andN-[3-(Chloromethyldimethylammonium)propyl]-3-methoxyestra-1,3,5(10)-trien-17β-amine chloride

Formic acid (97-98%, 47.5 ml) is added dropwise to a cooled and stirredmixture of estrone methyl ether (71.0 g) and 3-dimethylaminopropylamine(158 ml). The resulting mixture is heated in an oil bath (165°-175° C.)for 20 hours. The reaction is quenched in ice water (total volume 700ml) and the resulting alkaline (pH˜9) mixture is extracted twice withCH₂ Cl₂. The combined extracts are washed with dilute aq. NaOH and withbrine, dried (Na₂ SO₄), filtered, and concentrated. Hexane (250 ml) isadded to the residue and a white fluffy precipitate is filtered off. Thesolvent is removed, leaving a residual light brown oil which slowlycrystallizes. A portion (20 g) of this material is chromatographe oversilica gel (500 g) packed as a slurry in 7.5% of 10% NH₄ OH--CH₃ OH inCH₂ Cl₂. The column is eluted (300 ml fractions) with the same solventsystem. The desired material (about 14 g) is eluted in fractions 13-26an pooled in CH₂ Cl₂. As this solution stands at 20°-25°, crystals form.After two days, the crystals (4.803 g), mp 184°-192° C. with rapidheating, are collected by filtration. More crystals form in the filtrateand after 3 days these are collected. The compound can be recrystallizedfrom methanol-acetone in the freezer, giving the second title compoundas crystals, mp 184°-186° C. decomposition (dec) with bubbling.

The remaining reaction product is chromatographed over silica gel (1.5kg) packed as a slurry in 7.5% of 10% NH₄ OH--CH₃ OH in chloroform. Thecolumn is eluted (350 ml fractions) with the same solvent system.Fractions 28-39 are pooled, dissolved in acetone-CHCl₃ and crystallizedby adding hexane and cooling. Crystals are collected, mp 160°-→>225° C.The filtrate is concentrated to an oil which crystallizes. This is takenup in ether and ethereal HCl is added. A precipitate is collected andrecrystallized from methanol-i-propyl alcohol, giving crystals of thefirst title compound, mp 278°-283° C. dec. (PLA2)

EXAMPLE 2N-Methyl-N-[3-(dimethylamino)propyl]-3-methoxyestra-1,3,5(10)-trien-17.beta.-amine,methylene chloride adduct

A solution of 18 g of the title compound from Preparation 2 is allowedto stand for about 2 days in ˜300 mL of methylene chloride at 25°. TLCanalysis (15/85/2 methanol/methylene chloride/ammonium hydroxide) shows,in addition to the expected starting compound, a new much more polarcomponent which accounts for about one-third of the material. Followingremoval of the methylene chloride in vacuo, the residue is taken up in300 mL of ethyl acetate. The mixture is stirred at 20°-25° for 15 min,then diluted with 30 mL of hexane and stirred 10 min longer. The solidsare isolated via filtration through a medium-porosity sintered glassfunnel and washed with 200 mL more 90/10 ethyl acetate/hexane. Afterdrying for 1 hr at 25°, 0.1 mm, the title compound is obtained having amp 164°-165° (dec, mp varies with rate of heating). (PLA2, diabetes)

EXAMPLE 3 N-Methyl-N-[3-(methylamino)propyl[-3-methoxyestra-1,3,5(10)-trien-17β-amine andN-methyl-N-(2-propenyl)-3-methoxyestra-1,3,5(10)-trien-17β-amine

A solution of 469 mg of the title compound from Example 2 and 340 mg ofsodium ethoxide in 10 mL of ethanol is heated at reflux for 1 h, bywhich time TLC indicates that no starting material remains. Themixtureis filtered through a fine-porosity sintered glass funnel, andthe filtrate, with ethanol washes, is concentrated in vacuo. The residueis chromatographed on an 80 g column of silica gel which is previouslydeactivated with 8 mL of ammonium hydroxide. The column is packed andeluted (8 mL fractions) with 5/95/1 methano-l/methylenechloride/ammonium hydroxide.

Fractions 19-21 contain the second title compound which crystallizesupon trituration with methanol. Filtration and drying (0.1 mm, 16 h,25°) yields the compound, mp 88°-89° C.

Elution of the above chromatogram with 10:90:2 methanol/methylenechloride/ammonium hydroxide (fractions 47-58) affords the first titlecompound as an oil; IR νmax (neat) 1600, 1570, 1500, 1460, 1280, 1255,1240, 1150, 1035, 905 cm⁻¹. (PLA2) (2nd compound, PAA2-)

EXAMPLE 4N-[3-(Trimethylammonium)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17.beta.-amineiodide

A solution of the amine produced in Preparation 4 (193 mg) in methanol(10 ml) is treated at 20°-25° with methyl iodide (284 mg). The reactionis stirred at 20°-25° for 20 hours. A precipitate forms which iscollected by filtration to give the title compound, mp 206° C., dec.(PLA2)

EXAMPLE 5N-[3-(Dimethylbenzylammonium)propyl]-N-methyl-3-methoxyestra-1,3,5(10)-trien-17β-aminechloride

Following the general procedure of Example 2, but starting with benzylchloride, the title compound is produced, mp 199°-210° C. (PLA2)

Example 6 N-[3-(Dimethylcarbethoxymethylammonium)proply]-N-methyl-3-methoxyestra-1,3,5(10)-trien-17β-amine-chloride

Following the general procedure of Example 2, but starting with ethylchloroacetate, the title compound is produced, mp 159°-163° C. (PLA2)

EXAMPLE 7N-[3-(Dimethylamino)propyl]-N-methyl-3-methoxyestra-1,3,5(10)-trien-17.beta.-amine,N-mono-oxide and N,N'-dioxide

A solution of 4.7 g of the title compound from Preparation 2, and 5 mlof 30% aqueous hydrogen peroxide in 120 ml of methanol is stirred at20°-25° for 72 hr. Additional 5 ml portions of hydrogen peroxide areadded at 6 hr and 28 hr. After 72 hr, an argon atmosphere sphere isintroduced, and a suspension of 100 mg of 10% platinum/carbon in 1.5 mlof ethanol is added in one portion. The stirred mixture is cooledintermittently to moderate the (initially) fairly vigorous evolution ofoxygen. The mixture is stirred for 4 hr (oxygen evolution appearscomplete at about 2 hr) and then filtered through a pad of Celite on amedium porosity sintered glass funnel. The filtrate, including washeswith additional methanol, is evaporated to dryness on a rotaryevaporator behind a safely shield, and the residue is azeotroped withsix 200 ml portions of methylene chloride to remove most of the water.

A 500 mg portion of silica gel is shaken with 50 ml of concentratedaqueous ammonium hydroxide until the silica becomes homogeneous andfree-flowing. The ammonia-treated silica is allowed to stand overnightat 25° C. in a stoppered flask and is then slurrypacked into a columnwith 10/90/1 mthanol/methylene chloride/ammonium hydroxide. The columnis eluted with the same solvent (100-150 ml fractions).

The first title compound is obtained from fractions eluting between1500-2300 ml. Evaporation of these fractions affords an oil, which turnsto a solid upon trituration with 75 ml of ether. Filtration and drying(0.1 mm, 25°, 16 hr) yields the first title compound, a solid with mp78°-82° C. (PLA2, diabetes)

Elution of the above chromatogram with 50:50:2methanol/chloroform/ammonium hydroxide (10 L) yields the second titlecompound. After trituration with ether (˜100 ml), filtration, and drying(16 hr, 25° C., 0.01 mm), the solid exhibits a mp 94°-96° C. (PLA₂,diabetes)

The amount of hydrogen peroxide and the reaction time in this case arechosen intentionally to produce close to a 1:1 mixture of mono- andbis-oxide. At lower hydrogen peroxide concentration and shorter reactiontime the mono-oxide compound is the predominant product (˜70%), whilemore reagent and longer reaction time favors bis-oxide formation.

Example 8N-Methyl-N-(2-propenly)-3-methoxyestra-1.3.5(10)-trien-17β-amine

A 20 mg sample of mono-oxide compound prepared in Example 7 is heated at110° C. in the absence of solvent for 15 min. The material is thencooled to 20°-25° and chromatographed on a 10 g column of silica gelwhich is deactivated with 1 ml of concentrated ammonium hydroxide. Thecolumn is packed and eluted with 5:95:1 methanol/chloroform/ammoniumhydroxide (1 ml fractions). Fractions 15-18 yield the title compoundidentical by TLC to the material more fully characterized in Example 3.(PLA2-)

EXAMPLE 9N-[3-(Dimethylamino)propyl]-N-methyl-3-(1-phenyl-5-tetrazolyloxyestra-1,3,5(10)-trien-17β-amine

A degassed solution of the title compound from Preparation 11 (0.294 g)in acetonitrile (25 ml) is treated at 20°-25° under nitrogen with5-chloro-1-phenyl-1H-tetrazole (0.148 g) followed by anhydrous potassiumcarbonate (0.276 g). The resulting suspension is stirred at reflux for21 hours, permitted to cool to 20°-25° and concentrated in vacuo to alight brown residue.

The crude product is chromatographe on "flash chromatography"-3"×8.5" ofHPLC grade silica gel - in 1% ammonium hydroxide in 3:17methanol/methylene chloride while collecting ˜130 ml fractions.Fractions 4-10 are combined and concentrated to give the title compound.Further purification affords a solid, mp 204°-206° C. (PLA2)

EXAMPLE 10N-[3-(Dimethylamino)propyl]-N-formyl-estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Preparation 1, but starting with17-ketoestrane, the title compound is produced, mp 123°-125° C.

EXAMPLE 11N-[3-(Dimethylamino)propyl]-N-methylestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 10, the title compound is produced. IR 2950,2870, 2780, 1490, 1455, 1385, 1340, 1255, 1045, 745 cm₋₁. (PLA2)

EXAMPLE 12N-[3-(Dimethylamino)propyl]-N-methyl-3-acetoxyestra-1,3,5(10)-trien-17.beta.-amine

A solution of the title compound from Preparation 11 (0.218 g) inpyridine (3 ml) is cooled to 0° C. under nitrogen and treated withacetic anhydride (1.2 ml). The resulting solution is stirred at 0° C.and then permitted to warm to 20°-25° with stirring for 66 hours. Thereaction mixture is poured into ice-cold saturated sodium bicarbonate(150 ml) and extracted with ethyl acetate (3×120 ml). The combinedorganics are washed with aqueous saturated sodium bicarbonate, driedover sodium sulfate, filtered and concentrated to an oil. p The crudeproduct is chromatographed on "flash chromatography"-3"×81/2" column ofHPLC grade silica gel - in 1:10:90 -ammoniumhydroxide/methanol/methylene chloride while collecting 150 ml fractions.

Fractions 4-6 are combined and concentrated to give a solid, mp 40°-41°C. (PLA2)

EXAMPLE 13N-[3-(Dimethylamino)propyl]-N-methyl-3-butyroxyestra-1,3,5(10)-trien-17.beta.-amine

Following the general procedure of Example 12, but starting withn-butyric anhydride, the title compound is produced. IR (film) 2930,2860, 2815, 2765, 1755, 1605, 1495, 1455, 1375, 1345, 1225, 1155, 1075,1035, 1000, 935, 825 cm⁻¹. (PLA2)

EXAMPLE 144-Bromo-N-[3-(dimethylamino)propyl]-N-methyl-3-hydroxyestra-1,3,5(10)-trien-17β-amine

A solution of the title compound from Preparation 11 (0.43 g) inabsolute ethanol (30 ml) is treated at 20°-25° under nitrogen withN-bromo acetamide (0.24 g). The resulting yellow solution is stirred at20°-25° under nitrogen overnight and concentrated in vacuo.

The resulting yellow oil is chromatographed on two size B Lobar columns(in tandem) in 5% of a 9:1 methanol/ammonium hydroxide solution in 95%methylene chloride while collecting 18 ml fractions. Fractions 79-87 arecombined and concentrated to give the title compound, mp 75q20 -78° C.(PLA2)

EXAMPLE 15 N-[3-(Dimethylamino)propyl]-N-formyl-5α-androstan-17β-amine

Following the general procedure of Preparation 1, but starting with5αandrostan-17-one, the title compound is produced, mp 121.5°-122° C.(PLA2)

EXAMPLE 16 N-[3-(Dimethylamino)propyl]-N-methyl-5α-androstan-17β-amine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 15, the title compound is obtained. NMR(CDCl₃, TMS) δ 2.26, 0.80. (PLA2)

EXAMPLE 17N-Formyl-N-(3-methylbutyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 1, but starting withisoamylamine, the title compound is produced, mp 106°-107° C. (PLA2-,diabetes)

EXAMPLE 18N-Methyl-N-(3-methylbutyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 16, the title compound is produced, mp80°-80.5° C. (PLA2-, diabetes)

EXAMPLE 19N-Formyl-N-(3-hydroxypropyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 1but starting with3-amino-1-propanol, the title compound is produced, mp 159°-161° C.(PLA2-, diabetes-)

Example 20N-(3-Hydroxypropyl)-N-methyl-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 2, but starting with thetitle compound from Example 19, the title compound is produced, mpl121.5°-123.5° C. (PLA2)

Example 21N-(3-Ethoxypropyl)-N-formyl-3-methoxyestra-1,3,5(10)-triene-17β-amineand N-(3-Ethoxypropyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 1, but starting with3-ethoxypropylamine, the title compounds are produced. First titlecompound, mp 107.5°-108.5° C. (PLA2, diabetes -)

Second title compound: mp 64°-64.5° C. (PLA2, diabetes)

EXAMPLE 22N-(3-Ethoxypropyl)-N-methyl-3-methoxyestra-1,3,5(10)-triene-17β-amine

Following the general procedure of Preparation 2, but starting with thefirst title compound from Example 21, the title compound is obtained. IR(liquid film) 2970, 2934, 2866, 2853, 2780, 1611, 1501, 1466, 1454,1444, 1378, 1314, 1282, 1256, 1238, 1176, 1152, 1151, 1123, 1054, 1039cm⁻¹. (PLA2, diabetes)

EXAMPLE 23N-Formyl-N-[2-(2-hydroxyethoxy)ethyl]-3-methoxyestra-1,3,5(10)-triene-17.beta.-amine

Following the general procedure of Preparation 1, but starting with2-(2-aminoethoxy)ethanol, the title compound is produced, mp 134°-135°C. (PLA2)

EXAMPLE 24N-[2-(2-Hydroxyethoxy)ethyl]-N-methyl-3-methoxyestra-1,3,5(10)-triene-17.beta.-amine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 23, the title compound is produced, mp101.5°-102° C. (PLA2, diabetes)

EXAMPLE 25N-]2-(Dimethylamino)ethyl]-N-formyl-3-methoxyestra-1,3,5(10)-triene-17.beta.-amine

Following the general procedure of Preparation 1, but starting withunsym-dimethyl-aminoethylenediamine, the title compound is produced, mp98°-100° C. (PLA2, diabetes)

EXAMPLE 26N-[2-(Dimethylamino)ethyl]-N-methyl-3-methoxyestra-1,3,5(10)-triene-17.beta.-amineDisuccinate Salt

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 25, an oil is produced.

A portion of the oil (0.732 g) and succinic acid (0.469 g) are dissolvedin methanol (10 ml). Crystals form and are collected after two hours,giving a crystalline solid. Recrystallization from methanol gives thetitle compound, mp 162°-164° C. (PLA2, diabetes)

EXAMPLE 27N-[2-(Dimethylamino)ethyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amineDisuccinate Salt

Following the general procedure of Preparation 4, but starting with thetitle compound from Example 25, an oil is produced.

The oil (1.946 g) is dissolved in methanol (25 ml) and the solutioncombined with a solution of succinic acid (1.228 g) in methanol (25 ml).A finely divided, white precipitate forms and after cooling in therefrigerator, filtration of the mixture gives the title compound ascrystals, mp 147°-153° C. with slight yellowing. (PLA2, diabetes)

Example 28N-[4-(Dimethylamino)butyl]-3-methoxyestra-1,3,5(10)-trien-17β-aminedyhydrochloride

Following the general procedure of Example 33, but starting with4-dimethylaminobutylamine, an oil is produced.

The oil is redissolved in ether and ethereal HCl is added. The solventis removed under a stream of nitrogen, leaving a white solid.Crystallization from cold methanol gives a first crop, mp 280°-283° C.dec, and a second crop of crystals. Recrystallization from methanolgives the title compound, mp 284°-286° C. dec. (PLA2, diabetes)

EXAMPLE 29 (1'R orS)-N-(4'-Dimethylamino-1-methyl)butyl-3-methoxyestra-1,3,5(10)-trien-17.beta.-amine

Following the general procedure of Example 33, but starting with2-amino-5diethylaminopentane, the title compound is produced. IR 2965,2932, 2868, 2843, 2804, 1611, 1501, 1466, 1453, 1447, 1381, 1372, 1355,1336, 1314, 1304, 1282, 1256, 1237, 1202, 1152, 1133, 1101, 1070, 1053,1042 cm⁻¹. (PLA2)

EXAMPLE 30 17β-[1-(4-Methyl)piperzinyl[-3-methoxyestra-1,3,5-(10)-triene

Following the general procedure of Preparation 1, but starting withN-methylpiperazine, the title compound is produced, mp 154.4°-155.5° C.(PLA2, diabetes)

EXAMPLE 31 17β-[1-(4-Methyl)piperazinyl[-3-methoxyestra-2,5(10)-diene

Following the general procedure of Preparation 4, but starting with thetitle compound from Example 30, the title compound is produced, mp149°-151° C. (PLA2)

EXAMPLE 32N-[2-(N'-Morpholino)ethyl]-3-methoxyestra-1,3,5(10)-trien-17β-aminedihydrochloride

Following the general procedure of Example 33, but starting with2-(N-morpholino)ethylamine, a gummy, semi-solid precipitate is formed.When this mixture is acidified (pH ˜ 6-7) with aqueous HCl, a whitesolid precipitates. The solid is collected and air dried overnightgiving product. Recrystallization from methanol-ether gives colorlesscrystals, mp dec from 235°-280° C. A sample is recrystallized frommethanol-ether, giving the title compound as colorless crystals; mp, decfrom 250°-295° C. (PLA2, diabetes)

EXAMPLE 33N-[3-(N'Morpholino)propyl]-3-methoxyestra-1,3,5(10)-trien-17β-amine

3-(N'-morpholino)propylamine (24.0 g), glacial acetic acid (20 g),estrone methyl ether (9.5 g), NaB(CN)H₃ (2.09 g), and tetrahydrofuran(80 ml) are added sequentially to methanol (110 ml). The resultingmixture is stirred at 20°-25° for about 2 days, after which a clearsolution is seen. TLC (10% of a 1:3 NH₄ OH-CH₃ OH solution in CHCl₃ ;40% ethyl acetate in hexane) reveals that starting steroid is consumedand a major, new more polar material forms. Excess solvent is removedunder reduced pressure. Water (300 ml) is added to the residue. After 15minutes, this solution is washed with ether and then made alkaline bythe addition of 50% aqueous NaOH. A cloudy mixture results and isextracted with ether (100 ml). A second extraction with ether (150 ml)is done and, while in progress, crystals form in the first extract. Thefirst extract is cooled on ice and after several hours, the crystals arecollected, mp 112°-114° C. The filtrate and the second extractionsolution are combined, dried (MgSO₄), filtered, and reduced in volume to50 ml from which a second crop of crystals is collected, mp 110°-114° C.A sample of the first crop is recrystallized from ether, giving thetitle compound, mp 114°-116° C. (PLA2, (PLA2, diabetes)

EXAMPLE 34N-[2-(2'-Morpholinoethyl)aminoethyl]-3-methoxyestra-1,3,5(10)-trien-17.beta.-aminetrihydrochloride

Following the general procedure of Example 33, but starting withN-morpholinoethylenediamine, an oil is produced. The oil is redissolvedin ether and etheral HCl is added. The solvent is blown off with astream of nitrogen, leaving a solid. The solid is recrystallized fromwater-acetone, giving a white solid. A sample is recrystallized fromwater-acetone giving the title compound, mp 225°-240° C. dec. (PLA2,diabetes)

EXAMPLE 353-Methoxy-N-[3-(pyrrolidin-2-on-1-yl)propyl]estra-1,3,5(10)-trien-17β-amineacetate

Following the general procedure of Example 33, but starting with1-(3-aminopropyl)-2-pyrrolidinone, an oil is produced. The oil isdissolved in chloroform (100 ml). Acetic acid (2.5 ml) is added and thesolution is diluted with ether (400 ml) and crystallization allowed toproceed. After cooling on ice, colorless crystals are collected.Recyrstallization from chloroform-ether gives the title compound ascolorless crystals, mpl 113°-123° C. (PLA2, diabetes)

EXAMPLE 36N-[3-(Imidazoyl)propyl]-3-methoxyestra-1,3,5(10)-trien-17β-amine acetate

Following the general procedure of Example 35, but starting withN-(3-aminopropyl)imidazole, the title compound is produced, mp 82°-82°C. (PLA2, diabetes)

EXAMPLE 373-Methoxy-N-[2-(1-methylpyrrol-2-yl)ethyl]estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with2-(2-aminoethyl)-1-methylpyrrole, the title compound is produced, mp141°-143° C. (PLA2, diabetes)

EXAMPLE 38 N-(2-Furfuryl)-3-methoxyestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting withfurfurylamine, the title compound is produced; mp 60°-62° C. (PLA2,diabetes)

EXAMPLE 39 N-Cyclopentadecyl-N-(3-dimethylaminopropyl)-N-formylamine andN-cyclopentadecyl-N-(3-dimethylaminopropyl)amino, acetic acid salt

Following the general procedure of Preparation 1, but starting withcyclopentadecanone, the title compounds are produced. The first titlecompound is rechromatographed on 395 g of 40-63 μm silica gel with 5%triethylamine-ethyl acetate. The product is obtained in fractions 71-110as an oil. IR (liquid film) 2928, 2858, 2815, 2780, 2765, 2724,1674,1459, 1421, 1407, 1261, 1212 cm⁻¹. (PLA2)

The N-cyclopentadecyl-N-[3-(dimethylamino)propyl]amine isrechromatographed on 395 g of 40-63 μm silica gel using ethylacetate-methanol-triethylamine (85/10/5) for elution. The product isobtained in fraction 47-90 as an oil which crystallizes. This is thenrecrystallized from hexane to give the second title compound, mp 88°-90°C. (PLA2, diabetes)

EXAMPLE 40 N-Cyclopentyldecyl-N-[3-(dimethylamino)propyl]-N-methylamine

Following the general procedure of Preparation 2, but starting with thefirst title compound of Example 39, the title compound is produced. IR(liquid film) 2928, 2857, 2812, 2782, 1459, 1266, 1253, 1211, 1153,1123, 1097, 1076, 1062, 1043, 1033, 836, 710 cm⁻¹. (PLA2, diabetes-)

EXAMPLE 41 N-Cyclopentyldecyl-N-(3-dimethylaminopropyl)-N-methylamine,disuccinate salt

The diamine of Example 40 (274 mg) in methanol (3 ml) is treated withsuccinic acid (238 mg) in methanol (3 ml). The reaction is stirred at20°-25° for 18 hours. A white precipitate forms which is collected byfiltration. This is then recrystallized from methanoldiethyl ether togive a solid, mp 164°-166° C. Second crop mp 164°-165° C. (PLA2)

EXAMPLE 42 N-[3-Dimethylalmmoniumpropyl]-Cyclododecamne acetate

Following the general procedure of Example 33, but starting withcyclododecanone, a pale yellow oil (semi-solid) is produced. This oil ispartitioned between chloroform (300 ml) and water (600 ml) and adjustedto pH 12 with 2 M potassium hydroxide. The layers are separated, and theaqueous layer is extracted with chloroform (300 ml). The organics arewashed with 1 M potassium hydroxide (200 ml, with gentle shaking) and1:1 brine-water (250 ml), dried over sodium sulfate, filtered andconcentrated to a light yellow oil which becomes semi-solid upon storageunder high vacuum overnight.

The crude product is chromatographed on silica gel (1 kg) in 5%triethylamine in 5:1 ethyl acetate/95% ethanol while collecting 40 mlfractions. Fractions 247-364 are combined and concetrated to give asolid which is recrystallized from hot ethyl acetate-hexane to producethe title compound, mp 102°-6° C. (PLA2)

EXAMPLE 43 N,N-bis[3-(dimethylamino)propyl]-cyclododecamine

Following the general procedure of Example 33, but starting withN,N,N',N'-tetramethyl dipropylenetriamine and cyclododecanone, the titlecompound is produced. IR (mull) 2938, 2906, 2861, 2851, 2812, 2781,2762, 2724, 1469, 1460, 1445, 1266, 1249, 1043 cm⁻¹. (PLA2)

EXAMPLE 44N-[N',N'-Di-(2-aminoethyl)-2-aminoethyl]-N-cyclopentadecylamine

Following the general procedure of Example 33, but starting withtris-(2-aminoethyl)amine and cyclopentadecanone, the title compound isproduced; IR (liquid film) 3361, 3287, 2928, 2857, 2810, 1460, 1350,1282, 116, 1043, 893, 843, 711. (PLA2)

EXAKPLE 45 cis- andtrans-2-Cyclohexyl-N-[3-(dimethylamino)propyl]-1-aminocyclohexane

Following the general procedure of Example 33, but starting with3-dimethylaminopropylamine and 2-cyclohexyl cyclohexanone, the titlecompounds are produced. Cis: IR (liquid film) 3313, 2922, 2853, 2814,2785, 2764, 1458, 1448, 1376, 1268, 1174, 1169, 1162, 1152, 1110, 1098,1073, 1043, 892, 859, 847, 722, 687; trans: IR (liquid film) 3314, 2967,2924, 1575, 1461, 1448, 1373, 1295, 1265, 1169, 1152, 1135, 1120, 1073,1043, 1033, 893, 851. (PLA2)

EXAMPLE 46 cis- andtrans-4-Cyclohexyl-N-[3-(dimethylamino)propyl]-1-aminocycloheane

Following the general procedure of Example 45, but starting with4-cyclohexylcyclohexanone, the title compounds are produced. Cis: IR(liquid film) 3293, 2924, 2852, 2813, 2786, 2764, 1459, 1449, 1449,1377, 1359, 1267, 1171, 1042, 739 (PLA2); trans: IR (mull) 2818, 2787,2763, 2695, 2418, 2350, 2317, 2295, 1647, 1573, 1543, 1404, 1323, 1262,1042, 914, 651. (PLA2)

Example 47 cis- andtrans-N-(4-Aminobutyl)-4-cyclohexyl-1-aminocyclohexane

Following the general procedure of Example 46, but starting with1,4-diaminobutane, the title compounds are produced; cis: IR (liquidfilm) 3580 3292, 2922, 2851, 2807, 1576, 1472, 1449, 1379, 1359, 1331,1319, 1109, 889, 849, 820, 739 (PLA2); trans: IR (mull) 3660, 3421,3373, 3275, 3256, 3169, 3107, 1587, 1480, 1449, 1425, 1366, 1350, 1339,1315, 1134, 1127, 1119, 933, 915, 897, 889, 859, 847, 826, 802, 788,780, 741, 695. (PLA2) (trans, diabetes)

Example 48 cis- andtrans-N-[3-(Dimethylamino)propyl]-4-phenyl-1-aminocyclohexane

Following the general procedure of Example 46, but starting with3-dimethylaminopropylamine and 4-phenylcyclohexanone, the titlecompounds are produced; cis: IR (liquid film) 3294, 3161, 3081, 3061,3026, 2967, 2932, 2856, 2814, 2784, 2765, 2729, 1601, 1493, 1465, 1461,1450, 1375, 1358, 1267, 1262, 1179, 1170, 1157, 1129, 1098, 1070, 1042,1031, 1006, 997, 843, 755, 689 (PLA2); trans: IR (mull) 3380, 3265,3082, 3067, 3028, 2819, 2802, 2777, 1682, 1603, 1495, 1445, 1308, 1258,1175, 1117, 1105, 1031, 913, 892, 883, 755, 698, 659, 647. (PLA2)(trans, diabetes)

EXAMPLE 49 tarns-1-(3-Diethylaminopropyl)aminodecalin

Following the general procedure of Example 46, but starting withdiethylaminopropylamine and trans-1-decalone, the title compound isproduced. IR (liquid film) 3307, 2968, 2920, 2853, 2799, 1455, 1447,1383, 1369, 1201, 1164, 1159, 1128, 1105, 1085, 1070. (PLA 2, diabetes-)

EXAMPLE 50 trans-1-(3-Diethylaminopropyl)aminodecalin-dihydrochl ordidesalt

The title compound from Example 49 (100 mg) is dissolved in ether andtreated with an ether solution saturated with HCl gas. A white gummysubstance precipitates. The solvent is removed and the substancecrystallizes after a very thourough drying over P₂ O₅ in a vacuumdessicator. MP 156°-158.5° C. (PLA2)

Example 51 2-[N-(3-Dilmethylaminopropyl)]adamantanamine

Following the general procedure of Example 33, but starting with3-dimethylaminoipropylamine and 2-adamantanone, the title compound isproduced. TLC (ethyl acetate/methanol/triethylamine; 85:10:5) Rf=0.22;NMR (CDCl₃) δ 2.62, 2.32, 2.20, 2.10-1.20.

Example 52 2-[N-(3-dimethylaminopropyl)]adamantanamine, disuccinate salt

Succinic acid (1.18 g) is dissolved in methanol (10 ml). A solution ofthe title compound from Example 51 (944 mg) in methanol (5 ml) is addedto the succinic acid solution in one portion. The solution is stirred at20°-25° for 45 minutes. The methanonl is partially removed at a reducedpressure and acetonitrile (50 ml) and ether is added. The solutionbecomes cloudy and a white precipitate forms. The white solid iscollected by filtration and dried in a vacuum dessicator over P₂ O₅ togive the title compound. The product is recrystallized twice fromacetonitrile and has a mp 102°-104° C. (PLA2, diabetes-)

EXAMPLE 53 N-(3-Dimethylaminopropyl)-N-methyl-5α-cholestan-3ε-amine,disuccinate salt

Following the general procedure of Preparation 1, but starting with5α-cholestan-3-one, an oil is produced. This formamide product is amixture of 3α and 3β isomers by NMR evidence. The isomers do notseparate by column chromatography. NMR (CDCl₃) δ 8.20, 8.12, 2.22,0.99-0.81, 0.67.

The product (1.32 g) described above is reduced with lithium aluminumhydride (0.99 g) as described in Preparation 2. The product ischromatographed on two Merck Lobar® size B columns. The sample isapplied in CH₂ Cl₂ and eluted with NH₄ OH/MeOH/CH₂ Cl₂ (95/4.5/0.5).Fractions with a volume of 20 ml each are collected and the product isobtained in fraction numbers 90-128 to give 0.597 g.

The product described above (597 mg) is dissolved in methanol (6) and asolution of succinic acid (472 mg, 4 mmol) in warm methanol (5 ml) isadded. After two or three minutes a white precipitate forms. Stirring iscontinued for 72 hours. The precipitate is collected by filtration andwashed with methanol. The product is dried in a vacuum dessicator at20°-25° over P₂ O₅. The title compound is thus obtained which isrecrystallized from methanol and has a mp 180.5°-182° C. (PLA2)

EXAMPLE 54 [3-(Dimethylamino)propyl]-1-aminoemthylpyrene

Following the general procedure of Example 33, but starting with3-dimethylaminopropylamine and 1-pyrenecarboxaldehyde, the titlecompound is produced. IR (liquid film) 3296, 3040, 2967, 2940, 2857,2814, 2779, 2765, 1603, 1588, 1459, 1182, 1096, 846, 842, 828, 819, 756,710, 681. (PLA2)

EXAMPLE 55(1,1'-Biphenyl)-4-acetamide,N-(3-dimethylaminopropyl)-2-fluoro-α -methyl

Using flurbiprofen (15.71 g), thionyl chloride (50 ml) and then in thesecond step 3-dimethylaminopropylamine (30 ml) as described previously,the amide is prepared. The product is recrystallized twice from hexaneto give the title compound, mp 71.5°-72.5° C. (PLA2)

EXAMPLE 563-Cyclohexene-1-carboxamide,N-(3-dimethylaminopropyl)-3-ethyl-4-(4-methoxyphenyl)-2-methyl

3-Ethyl-4-(p-methoxyphenyl)-2-methyl-3-cyclohexene-1-carboxylic acid(8.23 g) is mixed with thionyl chloride (25 ml). The mixture is heatedat reflux temperature for 45 minutes and then cooled to 20°-25°. Theexcess thionyl chloride is removed under reduced pressure. Toluene isadded to the residue and evaporated at reduced pressure to aid in thethionyl chloride removal.

A solution of 3-dimethylaminopropylamine (12.18 g) in toluene (25 ml) istreated with a solution of acid chloride (prepared above) in toluence(50 ml). The reaction is stirred for 16 hrs at 20°-25°.

The reaction mixture is diluted with ethyl acetate and poured intowater. The pH is adjusted to ˜ 10. The layers are separated. The organiclayer is washed with half-saturated brine (2 x), dried (anhydrous N₂SO₄), filtered and evaporated. The residue is chromatographed on 63-200μm silica gel which is slurry packed with triethylamine/methanol/ethylacetate (5/10/85). Eluting with the same solvent, fractions whichcontain 350 ml each are collected. The crystalline product is collectedin fractions 10-16. The product is recrystallized twice from hexane togive the title compound, mp 91°-92° C. (PLA2)

EXAMPLE 57N-Formyl-3-methoxy-N-(3'pyridinyl)methylestra-1,3,5(10)-trien-17β-amineand 3-Methoxy-N-(3'-pyridinyl)methylestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Preparation 1, but starting with3-aminomethylpyridine, the title compounds are produced, the first titlecompound as colorless crystals. IR (mull) 1665, 1629, 1609, 1590, 1575,1495, 1255, 1151, 1041, 713 cm⁻¹. (PLA2-, diabetes)

The major portion of the fractions contains mixtures of the two productsand is rechromatographed over silica gel (750 g) packed in ethylacetate. Fractions of 300 ml are collected. Fractions 34-42 contain theless polar first title compound and are pooled and crystallized fromacetone-hexane. Fractions 43-47 (mixtures) are pooled. Fractions 48-57(mixtures) are pooled and crystallization attempted from acetone-hexane.Fractions 58-63 (containing traces of less polar component) are pooledand recrystallized from acetone-hexane, giving the second titlecompound. Fractions 64-76 are pooled and recrystallized fromacetone-hexane, also giving the second title compound, mp 125°-127° C.Additional second title compound is obtained by physically separatingcrystals from the mixtures of the two products. This material has a mp126°-128° C. A sample is recrystallized again from acetone-hexane andgives the second title compound as crystals, mp 127°-129° C. (PLA2-,diabetes)

EXAMPLE 583-Methoxy-N-(3'-pyridinylmehtyl)estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with3-aminomethylpyridine, the title compound is produced. (PLA2-, diabetes)

EXAMPLE 593-Methoxy-N-(2'-pyridinylmethyl)estra-1,3,5(10)-trien-17β-aminehydrochloride

Following the procedure of Example 33, but starting with2-(aminomethyl)pyridine, an oil is produced. The oil is redissolved inether and ethereal HCl is added. The solvent is blown off with a streamof N₂ leaving a solid. The solid is crystallized from water (200 ml).Recrystallization from hot water (300 ml) gives the title compound, mp125°-128° C. (PLA2, diabetes)

EXAMPLE 603-Methoxy-N-(4'-pyridinylmethyl)estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with4-aminomethylpyridine, the title compound is produced, mp 157°-159° C.(PLA2-, diabetes)

EXAMPLE 61 N-Benzyl-3-methoxyestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting withbenzylamine, the title compound is produced, mp 102°-104° C. (PLA2-,diabetes)

EXAMPLE 62 N-Formyl-3-methoxyestra-1,3,5(10)-trien-17β-amine

A mixture of estrone methyl ether (14.2 g) and 2-(2-aminoethyl)pyridine(24 ml, 24.4 g) is stirred and cooled in an ice bath. To this mixture,formic acid (97-98%, 11.5 ml) is added in portions. The mixture is thenheated in an oil bath (180°-190° C. bath temp.) for 24 hours. Themixture is poured onto ice, treated with NaOH, and extracted withmethylene chloride (3X). The extracts are dried (Na₂ SO₄), filtered, andconcentrated to give a solid. The solid is crystallized from methylenechloride-hexane to give crystals. Recrystallization from methylenechloride-acetone gives the title compound, mp 252°-253° C. preceded byyellowing. (PLA2-, diabetes)

EXAMPLE 633-Methoxy-N-[2-(2'-pyridinyl)ethyl]estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with2-(2-aminoethyl)pyridine, the title compound is produced; mp 130°-132°C. (PLA2-, diabetes)

Example 643-Methoxy-N-[{2-[N'-(5-nitro-2-pyridinyl)-amino]ethyl]{estra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with2-(2-aminoethylamino)-5-nitropyridine, the title compound is produced,mp 107°-109° C. (PLA2-, diabetes)

EXAMPLE 65 N-Cyclopentadecyl-N-(3-pyridylmethyl)amine

Following the general procedure of Example 33, but starting with3-aminomethylpyridine and cylcopentadecanone, the title compound isproduced, NMR (CDCl₃, TMS) δ 8.58, 7.72, 7.25, 3.76, 2.56, 1.32.

EXAMPLE 66 N-Cyclopentadecyl-N-(3-pyridylmethyl)amine, succinate salt

Succinic acid (885 mg) is dissolved in methanol (10 ml). A solution ofthe title compound from Example 65 (950 mg) in methanol (5 ml) is addedot the succinic acid solution in one portion. The solution is stirred at20°-25° for 45 minutes and then the methanol is partially removed byevaporation at a reduced pressure. Next, ether is added to the partiallyevaporated solution and a white precipitate forms. The solid iscollected by filtration. The solid is recrystallized from acetonitriletwice to yield the title compound, mp 148°-149° C. (PLA2, diabetes-)

EXAMPLE 67 cis- andtrans-4-Cyclohexyl-1-[(3-methylpyridyl)-amino]cyclohexane

Following the procedure of Example 33, but starting with3-aminomethylpyridine and 4-cyclohexylcyclohexanone, the title compoundsare produced; cis: IR (Liquid film) 2923, 2851, 1576, 1478, 1448, 1423,791, 787, 714, 630 cm⁻¹ (PLA2); trans: mp 65-67° C.; IR (mull) 3267,3033, 2815, 2782, 1583, 1575, 1504, 1423, 1364, 1133, 1030, 845, 790cm⁻¹. (PLA2) (trans, diabetes)

EXAMPLE 68 N-(3-Aminopropyl)-3-methoxyestra-1,3,5(10)-trien-17β-amineand N,N'-bis(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,3-propanediamine

A stirred mixture containing 17.04 g of estrone 3-methyl ether, 41.6 gof 1,3-propanediamine bis-hydrochloride, 3.75 g of sodiumcyanoborohydride, and 60 g of 3Å molecular sieves in 420 ml of methanoland 300 ml of tetrahydrofuran is heated at reflux for 26 h, thenrecooled to 25°. Celite (3 tablespoons) is added, and the mixture isfiltered through a medium-porosity sintered glass funnel. The solids arewashed with an additional 500 ml of 4:3 methanol/tetrahydrofuran, andthe combined filtrate is evaporated to dryness. The residue followingevaporation of the first filtrate is partitioned between 1000 ml ofwater (adjusted to pH 12 with sodium hydroxide) and 400 ml ofchloroform, and the aqueous layer is further extracted with two 300 mlportions of chloroform. The organic extracts are washed with water,dried over anhydrous sodium sulfate, and concentrated in vacuo.

The crude product is chromatographed on 1.6 kg of 40-60 μ silica gel,which is equilibrated with 10 1 of 50/45/5 methylene chloride/ethylacetate/triethylamine and then eluted with 16 1 of the same mixture (1×41, then 50 ml fractions). Subsequent elution with 20 1 of 25/75/5methanol/methylene chloride/triethylamine is done to remove the morepolar product.

Fractions 91-97 yield the dimer second title compound which contains5-10% of a faster moving impurity tentatively identified as the17α-isomer.

Fractions 98-220 afford the second title compound, a solid.Recrystallization of a small portion of this material from ethyl acetateaffords crystals with mp 125°-140°. (PLA2, diabetes-)

The broad mp range and the observation of an m/z 36 peak in some massspec scans of the second title compound suggest that it may containsmall amounts of the corresponding hydrochloride salt.

Continued elution of the above chromatogram (the first ˜12 1 of the25/75/5 MeOH/CH₂ /Et₃ N solvent) afford a semi-sold residue. Triturationwith 10 ml of ethyl acetate, followed by filtration and drying (1 h,20°-25°, 0.1 mm) yields a white solid with mp 145° (dec). NMR shows thatthis solid is an approximate 1:1 mixture of the first title compound andEt₃ N⁺ CH₂ ClCl⁻ (from reaction of the Et₃ N and CH₂ Cl₂ used forelution). Rechromatography of this solid on 300 g of silica gel (elutionwith 38/60/2 MeOH/CH₂ Cl₂ /NH₄ OH) affords the first title compound asan oil. The mother liquors from the above crystallization arerechromatographed in the same manner and yield additional compound;IR(neat) 1610, 1577, 1501, 1431, 1352, 1336, 1314, 1281, 1256, 1237, 1179,1161, 1152, 1040, 900, 872, 816, 786 cm⁻¹. (PLA2, diabetes)

EXAMPLE 69 N-(3-Aminopropyl)-3-methoxyestra- 1,3,5(10)-trien-17β-amine,dihydrochloride

A solution of 400 mg of the first title compound from Example 68 in 2 mlof methanol is treated with 5 ml of 0.5 M HCl/methanol (non-aqueous).Precipitation of the salt from the clear solution begins in about 5 min.After most of the product has crystallized, ether is added until thetotal volume is about 25 ml. The solids are isolated by filtrationthrough a fine-porosity sintered glass funnel and are dried for 28 h at25° (0.05 mm), thereby, affording the title compound, with mp 274°-276°C. (dec). (PLA2, diabetes)

EXAMPLE 70 N,N'-Bis(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,3,-propanediamine, disuccinate

A suspension of 448 mg of the second title compound from Example 68 in 5ml of methanol is diluted with methylene chloride until the solutionbecomes clear. Most of the methylene chloride is removed by gentalheating, and the hot solution is treated with a solution of 173 mg ofsuccinic acid in 3.5 ml of methanol. The mixture is allowed to coolgradually to 20°-25° as the product precipitates. Ether (15 ml) is addedto the stirred mixture, and, after 30 min, the product is isolated viafiltration through a fine-porosity sintered glass funnel. The solids arewashed with 3:1 ether/methanol and dried for 18 h at 25° (0.08 mm),thereby affording the title compound, mp 191°-193° C. (PLA2)

EXAMPLE 71 N-(4-Aminobutyl)-3-methoxyestra-1,3,5(10)-trien-17β-amine andN,N'-(1,4-butanediyl)bis[3-methoxyestra-1,3,5(10)-trien-17β-amine]

A 1000 ml round-bottomed, 3-necked flask, equipped with a magneticstirrer and a reflux condenser, is flame-dried and then cooled in anatmosphere of nitrogen. The flask is charged with 9.4 g of estrone3-methyl ether suspended in 165 ml of tetrahydrofuran and 230 ml ofmethanol. To this suspension is added 33 g of 3Å molecular sieves,followed by 25 g of 1,4-diaminobutane dihydrochloride. The pH of thesolution is adjusted to 5.5 with glacial acetic acid, and 2.1 g ofsodium cyanoborohydride is added in one portion. The reaction isrefluxed for 16 h.

The reaction mixture is cooled and filtered through Celite, and thesolids are washed with 700 ml of 57% methanol/tetrahydrofuran. Thefiltrate is concentrated in vacuo. The resulting solid is partitionedbetween chloroform and water (pH 12). The aqueous layer is extractedseveral times with chloroform. The combined organic layers are driedover magnesium sulfate and concentrated in vacuo.

The crude product is chromatographed on 170 g of 70-230 mesh silica gel.The column is packed and eluted with (67:33:2) methylenechloride/methanol/conc. ammonium hydroxide (10 ml fractions).

Fractions 25-50 are combined based on their TLC homogeneity and affordthe second title compound. Fractions 301-500 are combined based on theirTLC homogeneity and afford the first title compound; first titlecompound: mp 76°-78° (PLA2); second title compound: mp >300° C.; IR(mineral oil mull) 3494, 3352, 3235, 3035, 2813, 2769, 2710, 1615, 1507,1321, 1290, 1257, 1244, 1040, 844, 831 and 632 cm⁻¹. (PLA2)

EXAMPLE 72 N-[4-Aminobutyl]-3-methoxyestra-1,3,5(10)-trien-17β-amine,disuccinate

A 10 ml round-bottomed, 2-necked flask, equipped with a magnetic stirreris flame dried and then cooled in an atmospher of nitrogen. The flask ischarged with 100 mg of the first title compound from Example 71dissolved in 1 ml of methanol. Then a solution containing 66 mg ofsuccinic acid in 2 ml of methanol (warmed to dissolve) is added in oneportion. The reaction mixture is stirred for 1.5 h at 20°-25°. A whiteprecipitate results, and the reaction mixture is concentrated in vacuo.

The resulting white solid is recrystallized from methanol/ether, dried(25° C., 0.1 mm, 64 h) and affords the title compound, mp 141.5°-162.2°C. (PLA2)

EXAMPLE 73N,N'-(1,4-Butanediyl)-bis[3-methoxyestra-1,3,5(10)-trien-17β-amine]disuccinate

Following the general procedure of Example 72, but starting withN,N'-(1,4-butanediyl)bis[3-methoxyestra-1,3,5(10)-trien-17β-amine], thetitle compound is produced, mp 248.0°-253.0° C. (PLA2-)

EXAMPLE 74 N-(5-Aminopentyl)-3-methoxyestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting with1,5-diaminopentane, the title compound is produced; mp 125.0°-222.0°; IR(mineral oil mull) 3029, 2755, 2688, 2608, 2560, 1611, 1500, 1367, 1357,1350, 1336, 1314, 1283, 1280, 1259, 1248, 1238, 1153, 1036, and 814cm⁻¹. (PLA2)

EXAMPLE 75 N-(5-Aminopentyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine,dihydrochloride

A 25 ml round-bottomed; 2-necked flask, equipped with a magneticstirrer, is flame dried and then cooled in an atmosphere of nitrogen.The flask is charged with 606 mg of the title compound from Example 74dissolved in 10 ml of methanol. To this solution (methanol) in oneportion. The reaction mixture is stirred for 2 h at 20°-25°.

The reaction mixture is diluted with 150 ml of diethyl ether, theprecipitate is filtered, dried (25° C., 0.1 mm, 16 h) and affords thetitle compound; mp 135.6° C. (dec). (PLA2)

EXAMPLE 76 N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-1,6-hexanediamineand the corresponding dihydrochloried salt

Following the general procedure of Example 33, but starting with1,6-hexanediamine, the title compound is produced, mp 93°-94° C. (PLA2)

The dihydrochloride salt is made by dissolving the title compound (52.2mg) in 0.5 ml of methanol and adding 0.56 ml of 0.5 M hydrogen chloridein methanol. The solution is stirred for 10 minutes at 20°-25°. Additionof diethyl ether is carrie out dropwise until the cloud point is reachedat which time a white precipitate forms. The solid is filtered, washedwith diethyl ether, and dried under high vacuum to provide thedihydrochloride salt, mp 231-237° C. (PLA2)

EXAMPLE 77 N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-1,8-octanediamine

Following the general procedure of Example 33, but starting with1,8-octanediamine, the title compound is produced, mp 47.5°-49.0° C.(PLA2)

EXAMPLE 78 N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-1,10-decanediamine

Following the general procedure of Example 33, but starting with1,10-decanediamine, the title compound is produced, mp 55.5°-57.5° C.(PLA2)

EXAMPLE 79N-(4-Amino-4-carbomethoxybutyl)-3-methoxyestra-1,3,5(10)-trien-17β-amineand 17-(3-Amino-[2-oxopiperidinyl)-3-methoxyestra-1,3,5(10)-triene

Following the general procedure of Example 33, but starting withornithine methyl ester, the title compounds are produced; first titlecompound: NMR (CDCl₃, TMS) δ 7.3-6.55, 3.75, 3.70 and 0.75 ppm (PLA2);second title compound: NMR (CDCl₃, TMS) δ 7.4-6.6, 3.75 and 0.75 ppm.(PLA2-)

EXAMPLE 80N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-N'-dodecyl-1,3-propanediamineand the corresponding dihydrochloride salt

Following the general procedure of Example 33, but starting withN-dodecyl-1,3-propanediamine, the first title compound is produced; NMR(CDCl₃, TMS) δ 7.37-7.17, 6.87-6.63, 3.78, 3.0-2.53, 2.43-1.1, 1.0-0.8,0.75. (PLA2-)

The dihydrochloride salt is made by dissolving the first title compoundin methanol and adding 0.5 ml of 0.5M anhydrous hydrogen chloride inmethanol to produce a solid, mp 217°-221° C.

EXAMPLE 81N-Acetyl-N'-(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,6-hexanediamine

The starting diamine from Example 76 (0.142 g) is dissolved in 5 ml ofmethanol and the mixture cooled in an ice bath. With continued cooling,acetic anhydride (63.2 mg) is added to the above mixture, and stirringis continued for 35 minutes, at which time TLC indicates the reaction iscomplete. The solution is made basic with 2 M sodium hydroxide to a pHof 13-14. The solution is extracted with methylene chloride (4 X) andthe organic phase washed with water and brine, dried (Na₂ SO₄),filtered, and concentrated to give 150 mg of an oil. In an identicalmanner a smaller amount of the starting diamine (52.9 mg) and aceticanhydride (22 mg) are reacted to give upon workup 52.8 mg of an oil.

The above oils are combined and chromatographed on 25 g of 40-60 μsilica gel eluting with methylene chloride containing 6% of amethanonl-ammonium hydroxide (9:1) mixture. An initial fraction of 50 mlis collected followed by 2 ml fractions. Fractions 51-105 arehomogeneous by TLC and are combined and concentrated to provide thetitle compound; IR νmax (neat) 3291, 2928, 2860, 1652, 1609, 1573, 1556,1501, 1464, 1453, 1447, 1435, 1369, 1282, 1256, 1237,1151, 1040, 734 and726 cm⁻¹. (PLA2)

EXAMPLE 82N-[6-(Formylamino)hexyl]-3-methoxy-1,3,5(10)-estratrien-17β-amine

All glassware used in this reaction is flame dried under nitrogen. Theprocedure used is based on work by M. Waki and J. Meienhofer, J. Org.Chem., 42, 2019 (1977). A two-necked round bottomed flask, equipped witha nitrogen inlet tube and magnetic stir bar is charged with1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (0.338 g) and 2ml of chloroform. The mixture is cooled in anice bath and formic acid (74.6 mg) in 2 ml of chloroform is addeddropwise. After stirring the above mixture for 10 minutes, the contentsare added to a constant pressure addition funnel. Thecarbodiimide-formic acid solution is added to an ice-bath-cooledsolution of the starting diamine from Example 76 (0.153 g) in 2 ml ofpyridine. The entire reaction mixture is stirred for 41/2 hours withcooling in an ice bath. By TLC, the reaction is not complete and isallowed to warm to 20°-25° for 30 minutes prior to workup.

The reaction contents are poured into a methylen chloride:ethyl acetate(1:1) mixture and water is added. The water layer is adjusted to a pH of10, and the layers are separated. The water layer is adjusted to pH 13an extracted with a fresh portion of methylene chloride-ethyl acetate(1:1). The combined organic layers are washed several times with brine,but fail to remove a suspended solid in the organic layer. Afterallowing the organic phase to sit in the refrigerator overnight, a whitesolid settles to the bottom of the flask. The solution is decanted fromthe flask without transferring the solid (the solid is water soluble).The organic phase is washed with water and brine, dried (Na₂ SO₄),filtered, and concentrated to give an oil. In a similar manner acomparable amount of the starting diamine (0.204 g) is reacted to formthe formamide and gives an oil.

The above oils are combined and chromatographed on 20 g of 40-60 μmsilica gel eluting with methylene chloride containing 5% of amethanol-ammonium hydroxide (9:1) mixture. An initial fraction of 80 mlis collected followed by 2 ml fractions. Fractions 46-80 contain thedesired product along with a minor impurity of higher Rf. The fractionsare combined and concentrated to give the title compound as a solid; IRνmax (mull) 3283, 1667, 1609, 1576, 1537, 1501, 1354, 1312, 1281, 1255,1237, 1151, 1132, 1039, 816, 780 and 710 cm⁻¹. (PLA2)

EXAMPLE 83N-[6-(Ethoxycarbonylamino)hexyl]-3-methoxy-1,3,5(10)-estratrien-17β-amine

A flame-dried 10 ml round-bottomed flask, equipped with a magnetic stirbar and nitrogen inlet tube is charged with the starting diamine fromExample 76 (0.151 g) in 3 ml of methylene chloride. The mixture iscooled in an ice bath and triethylamine (45.6 mg) in 1.5 ml of methylenechloride, followed by ethyl chloroformate (47.0 mg) in 1.5 ml ofmethylene chloride is added. The mixture is stirred for 30 minutes withcooling (by TLC the reaction is complete after 15 minutes). The contentsare poured into water and extracted with methylene chloride (3 X). Thecombined organic phase is washed with water and brine, dried (Na₂ SO₄),filtered, and concentrated to give an oil. In an identical manner asmaller amount of the starting diamine (42.2 mg) is reacted withtriethylamine and ethyl chloroformate to give an oil.

The above oils are combined and chromatographed on 25 g of 40-60 μsilica gel eluting with methylene chloride containing 5% of amethanol-ammonium hydroxide (9:1) mixture. An initial fraction of 80 mlis collected followed by 2 ml fractions. Fractions 6-110 are homogeneousby TLC and are combined and concentrated to provide the title compoundas a solid; IR νmax (neat) 3339, 2976, 2930, 2860, 2814, 1722, 1706,1610, 1516, 1501, 1464, 1452, 1313, 1304, 1280, 1256, 1239, 1151, 1134,1122, 1105, 1040 and 778 cm⁻³¹ 1. (PLA2)

EXAMPLE 84N-[6-(2,5-Dimethylpyrrolyl)hexyl]-3-methoxy-1,3,5(10)-estratrien-17β-amine

The procedure used is based on work by S. Breukelman, G. Meakins, and M.Tire, J.C.S. Chem. Commun., 800 (1982). The starting diamine fromExample 76 (0.139 g) is dissolved in 2.5 ml of benzene. To this solutionis added 2,5-hexanedione (75.5 mg) in 2.5 ml of benzene and acetic acid(16.4 mg) in 1 ml of benzene. The mixture is heated to reflux for 10minutes and then cooled (by TLC the reaction is complete by the time themixture achieves reflux temperature). Water is added to the mixture, andthe water layer is made basic with 2M sodium hydroxide to a pH of 13-14.The entire mixture is extracted with methylene chloride (4 X) and theorganic phase washed with water and brine, dried (Na₂ SO₄), filtered,and concentrateed to give an oil.

The above oil is chromatographed on 25 g of 40-60 μ silica gel, elutingwith methylene chloride containing 2.5% of a methanol-ammonium hydroxide(9:1) mixture. An initial fraction of 40 ml is collected followed by 2ml fractions. Fractions 31-117 are homogeneous by TLS and are combinedand concentrated to give an oil; IR νmax (neat) 2929, 2856, 2817, 1610,1518, 1501, 1464, 1454, 1409, 1394, 1313, 1300, 1281, 1256, 1237, 1151,1040, 816, 745 and 725 cm⁻¹. (PLA2)

EXAMPLE 85N,N',N'-Tris(methoxycarbonylmethyl)-N-(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,3-propanediamine

A solution of 342 mg of diamine from Example 68, 0.8 ml ofdiisopropylethylamine, and 0.28 ml of methyl bromoacetate in 20 ml oftetrahydrofuran is stirred at 20°-25° for two weeks. The mixture is thenpoured into 100 ml of 1:1 water/brine and extracted with two 100 mlportions of ethyl acetate. The extracts are washed with brine, driedover sodium sulfate, and concentrated in vacuo.

The crude product is chromatographed on a 75 g column of silica gel. Thecolumn is packed and eluted (7 ml fractions) with 40% ethylacetate/hexane. Fractions 59-100, clean by TLC, are combined and yieldthe title compound, an oil; IR νmax (neat) 1740, 1602, 1570, 1500, 1430,1280, 1260, 1240, 1200, 1170, 1020, 960 and 870 cm⁻¹. (PLA2-)

EXAMPLE 86N,N',N'-Tris(carboxymethyl)-N-(3-methoxy-1,3,5(10)-estratrien-17-yl)-1,3-propanediamine,tris (potassium salt)

A solution of 100 mg (0.179 mmol) of the title compound from Example 85in 4.0 ml of methanol is treated wiht 0.537 ml of 1.0M aqueous potassiumhydroxide and the initially cloudy solution is stirred at 25° for 20hours. (The mixture becomes clear by 30 min. ) Following removal of themethanol in vacuo, the residue is taken up in 5 ml of water andfreeze-dried, thereby affording the title compound; mp >325°; NMR (D₂ O,TMS) δ 7.3-6.5, 3.65, 3.5-3.0. (PLA2-)

EXAMPLE 87 3-Methoxy-N-methyl-estra-1,3,5(10)-trien-17β-amine and thecorresponding 17β-epimer

Following the general procedure of Example 33, but starting withmethylamine hydrochloride, the second title compound is produced; IRνmax (mull) 3309, 1608, 1503, 1478, 1448, 1445, 1433, 1310, 1289, 1252,1236, 1232, 1160, 1104, 1086, 1030, 888, 862, 824, 740 cm⁻¹. (PLA2,diabetes)

Continued elution of the large column above yields the first titlecompound. Recrystallization from 50 ml of ethyl acetate gives whiteneedles with mp 136°-137°. (PLA2, diabetes)

EXAMPLE 88N-[3-(3-Aminopropyl)amino]propyl-3-methoxyestra-1,3,5(10)-trien-17β-amineand the corresponding N,N'-dimer

Following the general procedure of Example 71, but starting with3,3'-iminobispropylamine, the title compounds are produced; first titlecompound: mp 53.2°-110.0° C.; IR νmax (mull) 3365, 3292, 3251, 3026,2821, 2759, 1609, 1581, 1501, 1367, 1359, 1313, 1287, 1255, 1236, 1155,1124, 1119, 1035, 817 and 786 cm⁻¹ (PLA2); second title compound: mp119.5-229.0° C.; IR νmax (mull) 3278, 3052, 3026, 2646, 1610, 1501,1351, 1314, 1282, 1256, 1237, 1160, 1152, 1033, 817 and 785 cm⁻¹. (PLA2)

EXAMPLE 89N-[3-(3-Amiopropyl)amino]propyl-3-methoxyestra-1,3,5(10)-trien-17β-amine,trisuccinate

A 25 ml round-bottomed, 2-necked flask, equipped with a magnetic stirreris flamed dried and then cooled in an atmosphere of nitrogen. The flaskis then charged with 500 mg of the first title compound from Example 88dissolved in 10 ml of methanol. Then a solution containing 443 mg ofsuccinic acid in 2 ml of methanol (warmed to dissolve) is added in oneportion. The reaction mixture is stirred for 1 h at 20°-25° (within 20min a solid precipitates).

The reaction mixture is diluted with 10 ml of diethyl ether, theprecipitate is filtered, dried (25° C., ˜ 10 mm, 100 h) and affords thetitle compound, mp 150.1°-153.0° C. (PLA2)

EXAMPLE 90N-[3-(3-Aminopropyl)amino]propyl-3-methoxyestra-1,3,5(10)-trien-17β-amine,trihydrochloride

Following the general procedure of Example 89, but starting withanhydrous hydrochloric acid solution, the title compound is produced; mp262.5°-267.5° C. (PLA2)

EXAMPLE 91N-(3-Methoxy)-1,3,5(10)-estratrien-17-yl)-N'-(3-di-methylamino)propyl-1,3-propanediamineand trihydrochloride salt

Following the general procedure of Example 33, but starting with thetriamine, the first title compound is produced; NMR (CDCl₃, TMS) δ7.33-7.17, 6.83-6.6, 3.73, 2.97-1.1, 2.2, 0.73. (PLA2)

The trihydrochloride salt is made by dissolving the first title compound(52 mg) in 0.5 ml of methanol and adding 0.75 ml of anhydrous 0.5Mhydrogen chloride in methanol. The solution is stirred for 10 minutes at20°-25°. Addition of diethyl ether is carried out dropwise until thecloud point of the solution is reached. A few additional drops of ethercause a white precipitate to form. The solid is filtered, washed withdiethyl ether, and dried under high vacuum to provide a powder, mp265°-268° C. (PLA2)

EXAMPLE 92 N-(3-Methoxy-1,3,5(10) -estratrien-17-yl)-diethylenetriamineand the corresponding trihydrochloride salt

Following the general procedure of Example 33, but starting withdiethylenetriamine, the title compound is produced; mp 120°-147° C.; IRνmax (mull) 3262, 3196, 3118, 3054, 1609, 1578, 1561, 1500, 1398, 1366,1353, 1338, 1312, 1286, 1252, 1237, 1032 and 816 cm⁻¹. (PLA2)

The trihydrochloride salt is prepared by standard methods, mp 180°-185°C. (PLA2)

EXAMPLE 93N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-1,4-bis(3-aminopropyl)piperazine

Following the general procedure of Example 33, but starting withy1,4-bis(3-aminopropyl)piperazine, the title compound is produced, mp62°-138° C.; IR νmax (mull) 3335, 3026, 2820, 2772, 1609, 1501, 1349,1315, 1286, 1254, 1237, 1152, 1147, 1034, 819 and 785 cm⁻¹. (PLA2)

EXAMPLE 94N-(5,9-Diazanonan-1-yl)-3-methoxyestra-1,3,5(10)-trien-17β-amine

Following the general procedure of Example 33, but starting withspermidine, the title compound is produced; IR νmax (neat) 2929, 2866,2849, 2816, 1664, 1610, 1576, 1501, 1466, 1453, 1436, 1314, 1281, 1256,1237, 1152, 1132, 1040, 816, 814, 784, 779 cm⁻¹. (PLA2)

EXAMPLE 95N-(5,9-Diazanonan-1-yl)-3-methoxy-estra-1,3,5(10)-trien-17β-aminetrihydrochloride

A solution of the title compound from Example 94, (0.17 g) in absolutemethanol (1 ml) is treated at 20°-25° under argon with 0.53M anhydrousHCl in methanol (2.8 ml) until acidic by pH paper. The resultingsolution is stirred for ˜ 5 minutes at 20°-25°, treated with anhydrousdiethyl ether (45 ml) over 30 minutes, and the resulting suspension isstirred for 15 minutes under argon. The suspension is filtered, and thefiltercake is washed with anhydrous ether (˜ 15 ml). The light yellowpowder is dried under high vacuum overnight to give the title compound,mp 230°-235° C. (PLA2)

EXAMPLE 963-Methoxy-N-(4,9,13-triazatridecan-1-yl)-estra-1,3,5(10)-trien-17β-amine

Followinhg the general procedure of Example 33, but starting withspermine, the title compound is produced; IR νmax (neat) 2928, 2865,2846, 2814, 1610, 1501, 1466, 1454, 1256, 1237, 1152, 1123, 1040, 817,785, 782 cm⁻¹. (PLA2)

EXAMPLE 973-Methoxy-N-(4,9,13-triazatridecan-1-yl)estra-1,3,5(10)-trien-17β-amine,tetrahydrochloride

A solution of the title compound from Example 96 (0.184 g) in absolutemethanol (1 ml) is treated at 20°-25° under nitrogen with 0.53Manhydrous HCl in methanol (3.1 ml) until acid by pH paper. Theresultingl solution becomes turbin within a minute and a precipitatebegins to form. The resulting suspension is stirred for ˜ 5 minutes, andanhydrous ether (45 ml) is added in portions over 30 minutes. Thesuspension is stirred for ˜ 15 minutes and filtered. The filtercake iswashed with anhydrous ether (˜ 15 ml) and the residue is dried underhigh vacuum to give the title compound; NMR (d₆ DMSO, TMS) δ 0.90,1.10-3.60, 3.70, 6.60-6.87, 7.23. (PLA2)

EXAMPLE 98N-Formyl-N',N'-dimethyl-N-[1-(3-methoxy-1,3,5(10)-estratrien-17-yl)ethyl]-1,3-propanediamine

Following the general procedure of Preparation 1, but starting with thetitle compound from Preparation 8 and 3-dimethylaminopropylamine, thetitle compound is produced; NMR (CDCl₃, TMS) δ 8.3-8.1, 7.33-7.13,6.87-6.63, 3.77, 3.6-2.63, 2.26, 2.43-0.6; TLC Rf=0.19 (more polarisomer) and 0.23 (less polar isomer), run in methylene chloride with 5%methanol-ammonium hydroxide (9:1), Rf=0.52 (more polar isomer) andRf=0.54 (less polar isomer), run in methylene chloride with 10%methanol-ammonium hydroxide (9:1).

EXAMPLE 99N,N',N'-Trimethyl-N-[1-(3-methoxy-1,3,5(10)-estratrien-17-yl)ethyl]-1,3-propanediamine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 98, the title compounds are produced; lesspolar isomer; NMR (CDCl₃, TMS) δ 7.37-7.2, 6.83-6.6, 3.77, 2.33, 2.12,3.0-1.07, 0.9-0.77, 0.72 (PLA2); more plar isomer: NMR (CDCl₃, TMS) δ7.37-7.17, 6.87-6.65, 3.8, 2.28, 2.17, 3.0-1.07, 1.0-0.83, 0.7. (PLA2)

EXAMPLE 100N-(N',N'-3-Dimethylamino)propYl-α-methyl-3-methoxy-1,3,5(10)-estratrien-17-methaneamine

Following the general procedure of Example 33, but starting with thefirst title compound of Preparation 8, the title compounds are produced;less polar isomer: NMR (CDCl₃, TMS) δ 7.33-7.2, 6.87-6.63, 3.87,3.4-3.1, 3.0-1.1, 2.23, 1.13-1.0, 0.73 (PLA2); more polar isomer: NMR(CDCl₃, TMS) δ 7.4-7.2, 6.87-6.67, 3.82, 3.0-1.1, 2.25, 1.22-1.08, 0.72.(PLA2)

EXAMPLE 1013-Methoxy-17-(3-dimethylamino-1-oxopropyl)-1,3,5(10)-estratriene

A 15 ml round-bottomed flask, equipped with a magnetic stir bar,condenser, and nitrogen inlet tube, is charged with the first titlecompound from Preparation 8 (0.454 g), dimethylamine hydrochloride(0.408 g), paraformaldehyde (0.201 g), 6 ml of absolute ethanol, and 90μl of 0.5M anhydrous hydrogen chloride in methanol. The above mixture isheated to 80° C. for 52 hours. By TLC, a large amount of startingmaterial is still present. The solution is cooled and additionaldimethylamine hydrochloride (0.134 g) and paraformaldehyde (97 mg) areadded. The mixture is again heated to 80° C. overnight. Analysis by TLCindicates starting material is still present, but enough product ispresent for workup. After cooling, the solution is made basic usingaqueous 2M sodium hydroxide. The mixture is extracted with methylenechloride (3X) and the organic phase washed with water and brine, dried(Na₂ SO₄), filtered, and concentrated to give 541 mg of an oil.

The oil is chromatographed on 60 g of 40-60 μ silica gel eluting with 2%triethylamine/ethyl acetate. An initial fraction of 75 ml is collected,followed by 4 ml fractions. Fractions 61-90 are nearly homogeneous byTLC and are combined and concentrated to provide a solid, mp 78°-82° C.(PLA2)

EXAMPLE 102 3-Methoxyestra-1,3,5(10)-trien-17β-ol,N,N'-bis(t-butoxycarbonyl)ornithine ester

A 10 ml round-bottomed, 2-necked flask, equipped with a magnetic stirreris flame dried and then cooled in an atmosphere of nitrogen. The flaskis charged with 116 mg of N,N'-bis(t-butoxycarbonyl)ornithine(Preparation 9) dissolved in 3 ml of ethylene dichloride. To the stirredmixture is added 5 mg of N,N-dimethylaminopyridine, 100 mg of theappropriate 17β-alcohol and 148 mg of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toleunesulfonate. The reaction mixture is stirredat 40° C. for 16 h.

The reaction mixture is cooled and then washed 4×70 ml with water. Theorganic layer is separated, washed with brine, dried over magnesiumsulfate and concentrated in vacuo.

The crude product is chromatographed on 40 gl of 230-400 mesh silicagel. The column is packed and eluted with 60% hexane/methyl ethyl ketone(2 ml fractions).

Fractions 30-42 are combined based on their TLC homogeneity and affordthe title compound; NMR (CDCl₃, TMS) δ 7.4-6.6, 3.75, 1.5 and 0.8 ppm.

EXAMPLE 103 3-Methoxyestra-1,3,5(10)-trien-17β-ol, ornithine esterbis(trifluoroacetate)

A 25 ml round-bottomed, 2-necked flasks, equipped with a magneticstirrer, is flame dried and then cooled in an atmosphere of nitrogen.The flask is charged with 75 mg of di(BOC) ester dissolved in 8.3 ml ofmethylene chloride and cooled to 0° C. (ice/water bath). To the stirredsolution is added 4.6 g of trifluroroacetic acid. The mixture is stirredfor 20 min at 0° C.

The reaction mixture is then concentrated in vacuo. The resultingresidue is triturated with ether and affords the title compound.Recrystallization from chloroform/hexane affords pure material, whichdecomposes between 175°-180° C.; NMR (CD₃ OD, TMS) δ 7.3-6.55, 3.75 and0.95 ppm. (PLA2)

EXAMPLE 104 3-Methoxyestra-1,3,5(10)-trien-17β-ol,3-(dimethylamino)propyl ether, methane sulfonate

A degassed solution of 3-methoxyestra-1,3,5(10)-trien-17β-ol (0.10 g) infreshly distilled THF (2 ml) is added at 20°-25° under N₂ to asuspension of 50% NaH (0.05 g) in THF (2 ml). The resulting suspensionis stirred for 2 h at 25° and treated with dimethyltrimethyleneammoniummethanesulfonate (0.38 g) (see Preparation 9). The resulting suspensionis stirred at reflux overnight with the reaction going ˜60-65%. Thereaction is permitted to go an additional 24 h, permitted to cool to20°-25° and is partitioned between ice-cold, saturated, aqueous sodiumbicarbonate (100 ml) and chloroform (100 ml). The layers are separated,and the aqueous layer is extracted with chloroform (2×100 ml). Theorganics are washed with aqueous saturated sodium bicarbonate (100 ml),dried over anhydrous potassium carbonate, filtered and concentrated togive crude product.

The crude product is chromatographed on silica gel (45) in 5% ethylacetate/hexane while collecting 10 ml fractions. Fractions 10-12 arecombined and concentrated to give starting material. Fractions 18-35 arecombined and concentrated to give the title compound; IR νmax (mull)2930, 2855, 2850, 2745, 1601, 1574, 1495, 1460, 1440, 1370, 1350, 1302,1275, 1245, 1225, 1130, 1085, 1025, 940 cm⁻¹.

EXAMPLE 105N-[3-Dimethylamino)propyl]-3-methoxyestra-1,3,5(10)-trien-17α-amine

A solution of 2.0 g (7.0 mmol) of 3-methoxyestra-1,3,5(10)-trien-17β-olin 35 ml of methylene chloride is cooled, under nitrogen, to 0° andtreatead with 1.46 ml of triethylamine, followed by 0.606 ml ofmethanesulfonyl chloride, the latter added dropwise over about 30seconds. The mixture is stirred at 0° for 30 min, by which time TLCanalysis (5% acetone/methylene chloride) shows the absence of startingalcohol and the presence of a single faster moving spot. Ice chips (˜5g) are added to the reaction mixture and stirring is continued for 15min at 0°. The reaction mixture is then diluted with 100 ml of methylenechloride and washed with cold dilute aqueous potassium bisulfate (2×50ml), followed by a single water wash. The combined organic layer isdried over anhydrous sodium sulfate and evaporated to dryness. The crudemesylate, a solid, is homogeneous by TLC and is used in the next stepwithout purification.

The crude mesylate is dissolved in 20 ml of freshly distilledN,N-dimethyl-1,3-propanediamine (bp 129°-130° at atmospheric pressure),and the mixture is heated at reflux in an atmosphere of nitrogen for 12days. The reaction mixture is cooled to 20°-25° and concentrated invacuo. The residue is diluted with water, adjusted to pH 12-13, andextracted with chloroform. The extracts are washed with water, driedover anhydrous sodium sulfate, and evaporated to dryness.

The crude product containing a small amount of the desired 17α-amine ischromatographed on a column containing 80 g of silica gel. Elution with300 ml of 5% acetone/methylene chloride yields an oil,3-methoxyestra-1,3,5(10),16-tetraene; IR νmax (neat) 3025, 1602, 1500,1445, 1425, 1280, 1260, 1240, 1040 cm⁻¹ ; NMR (CDCl₃, TMS) δ 7.4-6.6,6.1-5.4, 3.80, 0.78 ppm.

Elution of the above chromatogram with 15/85/1.5 methanol/methylenechloride/ammonium hydroxide gave impure title compound.

This material is rechromatographed on 80 g of silica gel, eluting with25/75/5 methanol/ethyl acetate/triethylamine (8 ml fractions). Fractions37-55 are combined and yield pure title compound which crystallizes onstanding. Recrystallization from ethyl acetate affords crystals with mp164°-168°. (PLA2)

EXAMPLE 106N-[3-Dimethylamino)propyl]-3-methoxyestra-1,3,5(10)-trien-17α-amine,disuccinate

To a stirred solution of 240 mg of the title compound from Example 105in 2 ml of methanol is added a warm solution of 153 mg of succinic acidin 3 ml of methanol. The homogeneous mixture is stirred for 20 min at25°, and is then concentrated to about one-third of the original volumeon the rotary evaporator. The crystalline product is recrystallized frommethanol/ether, and the solids are filtered, washed with 60/40ether/methanol and dried (0.08 mm, 2 h, 25°) to produce the titlecompound, mp 160.5°-161.0° C. (PLA2)

EXAMPLE 107N-[3-Dimethylamine)propyl]-N-formyl-11β-hydroxy-5α-androstan-17.beta.-amine

Following the general procedure of Preparation 1, but starting with11β-hydroxy-5α-androstan-17-one, the title compound is produced, mp142°-144° C.; NMR (CDCl₃, TMS) 0.95, 1.03, 1.04-2.45, 3.32, 4.30, 8.15.

EXAMPLE 108N-[3-(Dimethylamino)propyl]-N-methyl-11β-hydroxy-5α-androstan-17β-amine

Following the general procedure of Preparation 2, but starting with thetitle compound of Example 107, the title compound is produced, mp130°-131° C. (PLA2)

EXAMPLE 109 N-(3-Methoxy-1,3,5(10)-estratrien-17-yl)-N-methylacrylamide

A 25 ml two-neck round bottomed flask, equipped with a magnetic stirbar, nitrogen inlet tube, and 10 ml addition funnel is charged with thefirst title compound of Example 87 (1.0 g), triethylamine (0.40 g), and5 ml of methylene chloride. Dropwise addition of acryloyl chloride(0.335 g) in 5 ml of methylene chloride to the above solution isfollowed by stirring at 20°-25° for 30 minutes. Additional methylenechloride is added to the reaction mixture and the organic solution iswashed with water and brine, dried (Na₂ SO₄), filtered, and concentratedto provide a solid.

The solid is chromatographed on 140 g of 70-230 mesh silica gel elutingwith 15% ethyl acetate/methylene chloride. An initial fraction of 200 mlis collected, followed by 10 ml fractions. Fractions 48-95 arehomogeneous by TLC and are combined and concentrated to provide thetitle comound as a solid; mp 130°-132° C. (PLA2-)

EXAMPLE 110 N-Cyclopentadecyl-1,3-propanediamine andN,N'-bis(cyclopentadecyl)-1,3-propanediamine

Following the general procedure of Example 71, but starting withcyclopentadecanone and 1,3-diaminopropane bis(hydrochloride), the secondtitle compound is produced, mp 242°-248°. (PLA2)

Continued elution of the chromatogram, fractions 49-78 afford the firsttitle compound; NMR (CDCl₃, TMS) δ 2.9-2.4, 1.40 and 1.7-1.2 ppm. (PLA2)

EXAMPLE 111 N,N'-Bis(cyclopentadecyl)-1,3-propanediamine,dihydrochloride

A solution of 500 mg of the second title compound from Example 110 in 5ml of methanol is treated with 5 ml of 0.5M HCl in methanol (prepared bybubbling HCl gas into methanol and then titrating the solution). Theclear solution is stirred for 20 min, and then ether is added untilsolids begin to precipitate. The mixture is stirred for 18 h at 20°-25°and then diluted with 20 ml more ether. The solids are filtered, washedwith ether and dried (0.05 mm, 18 h, 25°), thereby affording the titlecompound; mp 237°-240°. (PLA2)

EXAMPLE 112 N-Cyclopentadecyl-1,3-propanediamine, dihydrochloride

A solution of 564 mg of the first title compound from Example 110 in 5ml of methanol is treated with 10 ml of 0.5M HCl in methanol added inone portion. After 20 min, the stirred, clear solution is graduallydiluted with ether at the rate of 3 ml/min until about 75 ml is added.(Precipitation of the salt begins after the addition of about 10 ml ofether.) The suspension is stirred for 1 h at 25°, then filtered, and thesolids are washed with 10 ml of 1:1 methanol/ether and dried (0.1 mm,25°, 18 h). The title compound so produced exhibits mp 268°-270° (dec).

EXAMPLE 113 N-Cyclohexyl-N'-dodecyl-1,3-propanediamine

Following the general procedure of Example 33, but starting withN-dodecyl-1,3-propanediamine and cyclohexanone, the title compound isproduced; IR νmax (neat) 3292, 2925, 2854, 2810, 1466, 1450, 1367, 1130and 721 cm⁻¹. (PLA2)

EXAMPLE 114 Diphenylphosphoric acid, 3-(N,N-dimethylamino)propyl amide

A 25 ml round-bottomed, 2-necked flask, equipped with a magneticstirrer, is purged with nitrogen. The flask is charged with 1.34 g ofdiphenyl chlorophosphate and 10 ml of chloroform. The flask is thencooled to 0° C. (ice/water). 0.64 g of N,N-dimethyl-1,3-propanediamineis added in one portion and allowed to stir at 0° C. for 10 min. Then208 mg of calcium hydroxide is added in small portions over a 20 minperiod. Once the addition is complete, 430 μl of a 12.75M sodiumhydroxide solution (aq.) is added over 10 min period. The reactionmixture is allowed to stir for 30 min at 20°-25°.

The reaction mixture is then dried over magnesium sulfate andconcentrated in vacuo. The crude product is chromatographed on a columncontaining 40 g of 70-230 mesh silica gel. The column is packed andeluted with 75% ethyl acetate/methanol with 2% triethylamine (5 mlfractions).

Fractions 11-26 are combined based on their TLC homogeneity to affordthe title compound; IR νmax (neat) 3223, 2968, 2945, 2861, 2818, 2770,1591, 1490, 1461, 1456, 1258, 1222, 1196, 1163, 1114, 1070, 1026 and1007 cm⁻¹. (PLA2)

EXAMPLE 115 Tris[3-(N,N-dimethylamino)propyl]-phosphoric traimide

A solution of 1 ml (1.645 g) of phosphorous oxychloride in 50 ml ofether is added over a period of 20 min to a stirred, 0° solution of 4.05ml (3.29 g) of N,N-dimethyl-1,3-propanediamine in 50 ml of ether. (Avigorous reacton ensues and a voluminous white solid forms). Thereaction mixture is then allowed to warm to 20°-25° and stirring iscontinued 18 h longer.

The mixture is cooled to 0° and treated with ammonia gas bubbled intothe mixure via a pipet for about 10 min. The solids are filtered, washedwith two additional 20 ml portions of cold ether, and dried undervacuum, thereby affording a hygroscopic solid, mostly ammonium chloride.The filtrate from above is concentrated in vacuo and affords an oil; IRνmax (neat) 3223, 2814, 2764, 1447, 1226, 1183, 1154, 1098, 1065, 1043,1010, 973, 830 cm⁻¹. (PLA2)

The following compounds are prepared by first preparing the imine byrefluxing a toluene solution of the appropriate steroid and amine in thepresence of a small amount of p-toluenesulfonic acid over a waterseparator until water no longer evolves. The solvent is evaporated andthe resulting imine is reduced to the amine by the method of Example 33.

    ______________________________________                                        Example                                                                              Compound                M.P.                                           ______________________________________                                        116    17β-(Phenylamino)-androst-5-en-3β-                                                          128.5-130                                             ol hydrate                                                             117    3-Methoxy-17β-(phenylamino)-estra-                                                               138-139                                               1,3,5(10)-triene (diabetes)                                            118    3-Methoxy-17β-[(3-pyridinyl)amino]-estra-                                                        258-261                                               1,3,5(10)-triene (diabetes)                                            119    3-Methoxy-17β-[(4-chlorophenyl)amino]-                                                             137-138.5                                           estra-1,3,5(10)-triene (diabetes)                                      120    3-Methoxy-17β-[(4-methoxyphenyl)amino]-                                                          161-163                                               estra-1,3,5(10)-triene (diabetes)                                      121    3-Methoxy-17β-[((3-trifluoromethyl)                                                              113-120                                               phenyl)-amino]-estra-1,3,5(10)-triene                                         (diabetes)                                                             122    3-Methoxy-17β -[(4-methoxycarbonyl)                                                                183-184.5                                           phenylamino]-estra-1,3,5(10)-triene                                           (diabetes)                                                             ______________________________________                                    

The following compounds are prepared by starting with the title compoundof Example 61, and formaldehyde (Example 123) and acetaldehyde (Example124), and then reducing by the method of Example 33:

    ______________________________________                                        Example Compound               M.P.                                           ______________________________________                                        123     3-Methoxy-N-methyl-17β-[(phenylmethyl)                                                          149-150.5                                              amino]-estra-1,3,5(10)-triene                                                 (PLA2, diabetes)                                                      124     3-Methoxy-N-ethyl-17β-[(phenylmethyl)                                                           113.5-115                                              amino]-estra-1,3,5(10)-triene (diabetes)                              ______________________________________                                    

The following compounds are prepared by following the general procedureof Example 33. The starting amines are 3-(aminomethyl)pyridine (Examples125-27), benzylamine (Example 128), 2-aminomethylfuran (Example 129),4-chlorobenzylamine (Examples 130 and 134),4-(2-aminoethyl)benzenesulfonamide (Examples 131 and 139),2-thiophenemethylamine (Examples 132 and 138),3-(trifluoromethyl)benzylamine (Examples 133 and 135),4-methoxybenzylamine (Example 136), 4-(trifluoromethyl)benzylamine(Example 137), 4-(aminomethyl)benzenesulfonamide hydrochloride (Example140), 2-(aminomethyl)benzimidazole dihydrochloride hydrate (Example141), 2-(trifluoromethyl)benzylamine (Example 142), glycine methyl esterhydrochloride (Example 143), 4-fluorobenzylamine (Example 144),3,4-dichlorobenzylamine (Example 145), 2,4-dichlorobenzylamine (Example146), and 2-chlorobenzylamine (Example 147). The starting steroids are3β-hydroxy-5-androsten-17-one (Examples 125 and 128-33),3α-hydroxy-5α-androstan-17-one (Example 126), 11β-hydroxy-5α-androstan-17-one (Example 127), and estrone methyl ether (Examples134-47).

    ______________________________________                                        Example                                                                              Compound                M.P.                                           ______________________________________                                        125    17β-[(3-Pyridinylmethyl)amino]-                                                                  196-201                                               androst-5-en-3β-ol                                                126    17β-[(3-Pyridinylmethyl)amino]-                                                                  158-162                                               5α-androstan-3α-ol                                         127    17β-[(3-Pyridinylmethyl)amino]-                                                                    186-187.5                                           5α-androstan-11β-ol                                         128    17β-[(Phenylmethyl)amino]-androst-5-en-                                                          155.5-157.5                                           3β-ol Ethanol solvate (diabetes)                                  129    17β-[(2-Furylmethyl)amino]-androst-5-en-                                                         106-108                                               3β-ol                                                             130    17β-[(4-Chlorophenylmethyl)amino]-                                                               181-185                                               androst-5-en-3β-ol (diabetes)                                     131    17β-[(2-(4-Aminosulfonylphenyl)ethyl)                                                              218-219.5                                           amino]-androst-5-en-3β-ol (diabetes)                              132    17β -[(2-Thienylmethyl)amino]-androst-                                                             143-144.5                                           5-en-3β-ol hydrate                                                133    17β-[((3-Trifluoromethyl)phenylmethyl)                                                             101-103.5                                           amino]-androst-5-en-3β-ol                                         134    3-Methoxy-17β-[(4-chlorophenylmethyl)                                                            131.5-132.5                                           amino]-estra-1,3,5(10)-triene                                                 (PLA2-, diabetes)                                                      135    3-Methoxy-17β-[((3-trifluoromethyl)                                                                70-71.5                                             phenyl-methyl)amino]-estra-1,3,5(10)-                                         triene (PLA2, diabetes)                                                136    3-Methoxy-17β-[(4-methoxyphenylmethyl)                                                           117.5-118.5                                           amino]-estra-1,3,5(10)-triene                                                 (PLA2-, diabetes)                                                      137    3-Methoxy-17β-[((4-trifluoromethyl)                                                              83.5-85                                               phenylmethyl)-amino]-                                                         estra-1,3,5(10)-triene                                                        (PLA2, diabetes)                                                       138    3-Methoxy-17β-[(2-thienylmethyl)amino]-                                                          85.5-87                                               estra-1,3,5(10)-triene (PLA2-, diabetes)                               139    3-Methoxy-17β-[(2-(4-aminosulfonyl-                                                              157-158                                               phenyl)ethyl)-amino]-estra-1,3,5(10)-triene                                   (PLA2-, diabetes)                                                      140    3-Methoxy-17β-[((4-aminosulfonyl)                                                                189-192                                               phenylmethyl)-amino]-estra-1,3,5(10)-triene                                   (PLA2-, diabetes)                                                      141    3-Methoxy-17β-[((2-benzimidazolyl)                                                               192-196                                               methyl)amino]-estra-1,3,5(10)-                                                triene (diabetes)                                                      142    3-Methoxy-17β-[((2-trifluoromethyl)                                                              112.5-114                                             phenyl-methyl)amino]-estra-1,3,5(10)-                                         triene (diabetes)                                                      143    3-Methoxy-17β-[(methoxycarbonylmethyl)                                                           57.5-59.5                                             amino]-estra-1,3,5(10)-triene (diabetes)                               144    3-Methoxy-17β-[(4-fluorophenylmethyl)                                                            71.5-73.5                                             amino]-estra-1,3,5(10)-triene (diabetes)                               145    3-Methoxy-17β-[(3,4-dichlorophenyl-                                                              130.5-132.5                                           methyl)amino]-estra-1,3,5(10)-                                                triene (diabetes)                                                      146    3-Methoxy-17β-[(2,4-dichlorophenyl-                                                              102.5-104.5                                           methyl)amino]-estra-1,3,5(10)-                                                triene (diabetes)                                                      147    3-Methoxy-17β-[(2-chlorophenylmethyl)                                                            136-137                                               amino]-estra-1,3,5(10)-triene (diabetes)                               ______________________________________                                    

The following compounds are prepared by following the general procedurefor Example 33. If the starting material is other than estrone methylether, it is noted in parentheses following the compound name.

    ______________________________________                                        Example                                                                              Compound                M.P.                                           ______________________________________                                        148    3-Hydroxy-17β-[((3-trifluoromethyl)                                                              49-52°                                         phenylmethyl)-amino]-estra-1,3,5(10)-triene                                   tetrahydrofuran solvate (estrone)                                             (diabetes)                                                             149    3-Methoxy-17β-[(2-(4-chlorophenyl)ethyl)-                                                        122-124°                                       amino]-estra-1,3,5(10)-triene (diabetes)                               150    3-(2,3-dihydroxypropoxy)-17β-                                                                    123-133°                                       [(4-chlorophenyl-methyl)amino]-                                               estra-1,3,5(10)-triene (2,3-                                                  dihydroxypropyl estrone ether) (diabetes)                              151    3-Methoxy-17β-[(4-methoxycarbonyl-                                                               122-133°                                       phenylmethyl)-amino]-estra-1,3,5(10)-                                         triene (diabetes)                                                      152    3-Methoxy-17β-[(4-bromophenylmethyl)                                                             143-144.5°                                     amino]-estra-1,3,5(10)-triene (diabetes)                               153    3-Methoxy-17β-[(3-chlorophenylmethyl                                                             60-63°                                         amino]estra-1,3,5(10)-triene                                                                          (diabetes)                                     154    3-Methoxy-17β-[(3-phenylpropyl)amino]-                                                           85-90°                                         estra-1,3,5(10)-triene (diabetes)                                      155    3-Methoxy-17β-[(4-phenylbutyl)amino]-                                                            97-100°                                        estra-1,3,5(10)-triene (diabetes)                                      156    3-Methoxy-17β-[(4-methylphenylmethyl)                                                            102-106°                                       amino]-estra-1,3,5(10)-triene (diabetes)                               157    3-Methoxy-17β-[(1-phenylethyl)amino]-                                                            70-78°                                         estra-1,3,5(10)-triene (diabetes)                                      158    3-Methoxy-17β-[(2-(1,2-dihydro-2-                                                                130-131°                                       oxopyrid-1-yl)ethyl)amino]-estra-1,3,5(10)-                                   triene (diabetes)                                                      ______________________________________                                    

The following compounds are prepared by starting with the title compoundof Example 135, and formaldehyde (Example 159), acetaldehyde (Example160), propionaldehyde (Example 161), iso-valeraldehyde (Example 162),octylaldehyde (Example 163), and tetradecyl aldehyde (Example 164), andthen reducing by the method of Example 33:

    ______________________________________                                        Example                                                                              Compound                M.P.                                           ______________________________________                                        159    3-Methoxy-N-methyl-17β-[((3-trifluoro-                                                           112.5-114°                                     methyl)-phenylmethyl)amino]-                                                  estra-1,3,5(10)-triene                                                 160    3-Methoxy-N-ethyl-17β-[((3-trifluoro-                                                            80-87.5°                                       methyl)-phenylmethyl)amino]-                                                  estra-1,3,5(10)-triene (diabetes)                                      161    3-Methoxy-N-(propyl)-17β-[((3-trifluoro-                                                         91-92.5°                                       methyl)-phenylmethyl)amino]-                                                  estra-1,3,5(10)-triene (diabetes)                                      162    3-Methoxy-N-(3-methylbutyl)-17β-                                                                 92-102°                                        [((3-trifluoromethyl)phenylmethyl)amino]-                                     estra-1,3,5(10)-triene hydrochloride                                          (diabetes)                                                             163    3-Methoxy-N-(octyl)-17β-[((3-trifluoro-                                                          86-309°                                        methyl)-phenylmethyl)amino]-                                                  estra-1,3,5(10)-triene hydrochloride                                   164    3-Methoxy-N-(tetradecyl)-17β-                                                                    116-119°                                       [((3-trifluoromethyl)phenylmethyl)amino]-                                     estra-1,3,5(10)-triene                                                 ______________________________________                                    

The following compounds are prepared by starting with acetaldehyde andthe title compound of Example 132, Example 139, and Example 142,respectively, and then reducing by the method of Example 33:

    ______________________________________                                        Example                                                                              Compound                 M.P.                                          ______________________________________                                        165    3-Methoxy-N-ethyl-17β-[(4-chlorophenyl-                                                           93.5-95°                                      methyl)-amino]estra-1,3,5(10)-triene                                          (diabetes)                                                             166    3-Methoxy-N-ethyl-17β-[(2-(4-aminosulfonyl-                                                       125-133°                                      phenyl)ethyl)-amino]-estra-1,3,5(10)-triene                                   (diabetes)                                                             167    3-Methoxy-N-ethyl-17β-[((2-trifluoro-                                                             70.5-75°                                      methyl)-phenylmethyl)amino]-                                                  estra-1,3,5(10)-triene (diabetes)                                      ______________________________________                                    

The following compound is prepared by starting with the title compoundfrom Example 154 and hydrocinnamaldehyde and then reducing by the methodof Example 33:

    ______________________________________                                        Example                                                                              Compound                  M.P.                                         ______________________________________                                        168    3-Methoxy-N-(3-phenylpropyl)-17β-[(3-phenyl-                                                       85-90°                                       propyl)amino]-estra-1,3,5(10)-triene (diabetes)                        ______________________________________                                    

EXAMPLE 169N-[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,6-hexane-diamine

1,6-Diaminohexane was dissolved in 800 ml of methanol and the pH of thesolution was brought to 6.7 with glacial acetic acid. To this was addedestrone methyl ether and 800 ml of THF. NaCNBH₃ was then added and thesolution was heated to 61° C. and kept there overnight. Reactioncompletion was found by TLC. The solution was made basic with 2M NaOH(1300 ml) to a pH above 12. The solution was then extracted with CH₂ Cl₂(3×1000 ml) and the combined organic layers were washed with brine,dried (MgSO₄), filtered, and concentrated to give a yellow oil. Uponstanding in the refrigerator the material turned to a yellow whitesolid.

The material was passed through a pre-column of 70-230 mesh silica geland eluted with CHCl₃ containing 25% of a MeOH/NH₄ OH (9/1) mixture.After the solvent was evaporated, the resulting oil was chromatographedon 1840 g of 15-40 μm silica gel and eluted with CHCl₃ containing 13% ofa MeOH/NH₄ OH (9/1) mixture. 300 ml fractions were collected withfraction numbers 33-53 being homogeneous by TLC. Fraction numbers 30-32contained a small amount of the α-isomer and another impurity but wereadded to the main batch of fractions. Solvent evaporation andtrituration with hexane provided an off-white solid which was placedunder (0.1 mm) high vacuum for 2 hours to give the title compound, m.p.93°-94.5° C.

EXAMPLE 1701H-Pyrrole-2,5-dione,1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]

The diamine from Example 169 and maleic anhydride were added to a3-necked round-bottomed flask (2 liter) eqipped with a condenser,nitrogen inlet tube, thermometer with thermometer adapter, and magneticstir bar. Both starting materials were covered with 850 ml of o-xylene,heated to a solution temperature of 144° C. (reflux) and kept there for1 hour and 15 minutes. Reaction completion was determined by TLC. Thesolution was allowed to cool and diluted with 1200 ml of CHCl₃. Thexylene and chloroform were evaporated to give a light brown gum.

This gum was chromatographed on 1840 g of 15-40 μm silica gel elutingwith CHCl₃ containing 3% of a MeOH/NH₄ OH (9/1) mixture. Fractions of400 ml were collected with fractions 11-25 being homogenous by TLC andcorresponding to the desired product. Solvent evaporation andtrituration with hexane provided a light yellow solid. This solid wasplaced under high vacuum (0.2 mm) for 2 hours to give the titlecompound, m.p. 123°-124° C., as confirmed by NMR. (PLA2)

EXAMPLE 171 L-Ornithine,N5-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-,monopotassium salt

1.08 g of estrone methyl ether was slurried in 30 ml of THF and 40 ml ofmethanol. 6 g of 3Å sieves were added followed by 1.25 g of ornithinemethyl ester dihydrochloride dissolved in 20 ml of methanol. The pH wasadjusted (25% Et₃ N/MeOH) to 6.0 and then 237 mg of NaCNBH₃ was added inone portion and the reaction mixture was stirred for about 4 days.Reaction progress was monitored by TLC. The product was worked up byfiltering through celite, washing the solids with 210 ml of (4:3)MeOH/THF, evaporating under reduced pressure, and then dissolving thewhite solid in CH₂ Cl2 and washing it with 2M NaHSO₄ until the aqueouslayer became acidic (caution HCN). The aqueous acidic layer wasseparated, made basic with 3m KOH (pH 13), and extracted with CH₂ Cl₂.This was dried over MgSO₄, filtered and evaporated under reducedpressure to yield crude product. The entire crude was chromatographedusing 50 g of 230-400 mesh silica gel eluting with a CH₂ Cl₂ /MeOHsolution (10% NH₄ OH) (9:1), and collected in 3 ml fractions after a 50ml column volume was voided. Fractions 145-210 yielded the methyl ester.

16 mg of this methyl ester was dissolved in 2 ml of MeOH and 39 μl of a1.0M KOH solution were added. An additional 2 ml of water was added tothe stirred reaction and the reaction progress was monitored by TLCuntil completion. There followed a workup by evaporation under reducedpressure to produce the title compound. TLC: Rf 0.19 (90:9:1, CH₂ Cl₂/MeOH/NH₄ OH) (PLA2)

EXAMPLE 1721H-Pyrrole-2,5-dione,1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]henxyl],citrate

A flame dried 3-necked 15 ml round-bottomed flask, equipped with amagnetic stir bar, glass stoppers and nitrogen inlet tube, was chargedwith the title compound from Example 170 and about 3 ml of THF. To thiswas added citric acid monohydrate which had been completely dissolved in1.5 ml of MeOH. Stirring at 20°-25° was continued for 30 minutes atwhich time ether was added dropwise. At least 5 ml of ether was addedbefore a white solid appeared. An additional 2 ml of ether was added toensure complete recovery of the salt. The mixture was filtered through aF fist funnel to give a white solid. The solid was placed under highvacuum (0.2 mm, 20°-25°) for 4 hours to produce the title compound. MS(M+H)⁺ : 465.3111 (PLA2)

EXAMPLE 173 1H-Pyrrole-2,5-dione,1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] ,methandesulfonate

A flame dried 3-necked, 15 ml round-bottomed flask equipped with anitrogen inlet tube, magnetic stir bar and glass stoppers was chargedwith the title compound from Example 170 and 3.5 ml of THF. To this wasadded methanesulfonic acid in 2.5 ml of THF. After about 20 minutes ofstirring, a white precipitate formed. To ensure complete formation ofthe solid, 5 ml of ether was added and stirring was continued another 10minutes. The mixture was filtered through a fine porosity fritted funneland the solid washed with one portion of cold THF. This provided a whitepowder. The solid was placed under high vacuum overnight to produce thetitle compound, m.p. 200.5°-202.5° C. (PLA2)

EXAMPLE 174 1,3-Propanediamine,N-[3-[[(5α,17β)-androstan-17-yl]amino]propyl]-N,N',N'-trimethyl

0.53 g of 5α-androstan-17-one was slurried in 10 ml of THF and 15 ml ofMeOH. 2 g of 3Å sieves were then added followed by 0.5 g ofN,N,N'-trimethyl-N'-(3-aminopropy)-1,3-propanediamine. The pH waslowered to 6-6.5 with glacial acetic acid, 120 mg of NaCNBH₃ was addedand the mixture was refluxed for 7 hours. TLC (CH₂ Cl₂ /10% NH₄ OH inMeOH (4:1)) revealed product formation with some contaminants. Thereaction mixture was cooled, filtered through Celite wash solids with1:1 MeOH/THF, evaporated under reduced pressure, and diluted with CH₂Cl₂. The solution was then made basic with 3M KOH, washed with saturatedNaCl solution, dried over MgSO₄, filtered and evaporated under reducedpressure to yield crude product. The entire crude product waschromatographed on a medium pressure system comprising 230-700 meshsilica gel (160 g), eluting with CH₂ Cl₂ /10% NH.sub. 4 OH in MeOH(6:1). After 140 ml of column volume was voided, 5 ml fractions (#'s55-150) were collected. NMR confirmed that the title compound wasproduced. MS (M+H)⁺ : 432.4314 (PLA2)

EXAMPLE 175 Cholane-3,7,11-triol, 24-[[3-(dimethylamino)propyl]amino]

2 g of 3α,7α,12α-trihydroxy-5β-cholanic acid and 0.68 ml of Et₃ N weredissolved in 100 ml of DMF and then cooled to 0° C. Once cooled, 0.64 mlof isobutylchloroformate was added, the mixture was stirred for 15minutes, and then 0.57 ml of 3 -dimethylaminopropylamine in 5 ml of DMFwas added. The mixture was allowed to warm to 20°-25° and stirredovernight. TLC (CH₂ Cl₂ /10% NH₄ OH in MeOH (4:1)) revealed productformation. The product was worked up by washing the entire reactionmixture 3 times with water (pH 12-13), washing with saturated NaCl,drying over MgSO₄, filtering and evaporating under reduced pressure, toyield crude amide.

957 mg of the crude amide was dissolved in 100 ml of pyridine. To thiswas added about 2.1 ml of BSTFA followed by refluxing to yield product.The reactoin mixture was evaporated under high vacuum to remove alltraces of pyridine. 1.5 g of LiAlH₄ was slurried in 5 ml of THF. Thecrude tetra-TMS intermediate was added to this in a dropwise manner in 5ml of THF and allowed to stir for about 3 days. 45% KOH was addedcarefully until gassing ceased and the solution was then diluted withTHF/ether (300 ml). The Al salts were filtered off, dried over MgSO₄,filtered and evaporated under reduced pressure to yield crude product.IR revealed a trace of starting material. NMR (no TMS) revealed that thecrude product containing the title compound. The entire crude wasdissolved in 5-7 ml of MeOH and then 100 ml of 0.1N HCl was added andthe mixture was stirred at 20°-25°. After 1.5 hours TLC (CHCl₃ /25% NH₄OH in MeOH) revealed product formation. The slution was made basic withconcentrated NH₄ OH (pH 9-10) and the entire solution was placed underhigh vacuum. The resulting residue was chromatographed on 200 g of70-230 mesh silica gel, and eluted with CHCl₃ /25% NH₄ OH in MeOH (4:1).After 200 ml of column volume was collected 5 ml fractions (numbers105-260) were collected. NMR was consistent with the title compound,m.p. 68°-73° C. (PLA2)

EXAMPLE 176 1H-Pyrrole-2,5-dione,1-[3-[[3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)methylamino]propyl]methylamino]propyl]

1.0 g of the steriod amine N-(0.33-aminopropyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,3-propanediaminewas dissolved in 30 ml of CHCl₃. A solution containing 491 mg of maleicanhydride in 10 ml of THF was then added and the mixture was stirred at20°-25°. TLC (CHCl₃ /25% NH₄ OH in MeOH (4:1)) revealed productformation together with remaining starting material and the di-maleamicacid. Because starting material was present an additional 50 mg ofmaleic anhydride was added in 1 ml of THF and the mixture was stirredfor 30 minutes. Subsequent TLC analysis indicated no starting materialpresent. The reaction mixture was evaporated under reduced pressure toyield a crude product. The entire crude was chromatographed on 285 g of230-400 mesh silica gel and eluted with CHCl₃ /25% NH₄ OH in MeOH (4:1)(medium pressure column). After 500 ml of column volume was collected, 6ml fractions (numbers 50-82) were collected. NMR was consistent with thedesired maleamic acid intermediate. 217 mg of this maleamic acid and0.33 ml of a 37% aqueous solution of formaldehyde were added to 15 ml ofacetonitrile followed by 89 mg of sodium cyanoborohydride. The mixturewas stirred at room temperature. TLC revealed about 60% completion ofthe reaction. Therefore, an additional 0.33 ml of 37% aqueousformaldehyde solution and 89 mg of sodium cyanoborhydride were added andthe mixture was stirred at room temperature. TLC revealed the presenceof the next crude maleamic acid intermediate and therefore the entirereaction mixture was evaporated under reduced pressure, to yield crudemaleamic acid. This crude maleamic acid was chromatographed on 60 g of230-400 mesh silica gel, eluting with CHCl₃ /25% NH₄ OH in MeOH (4:1).After 90 mls of column volume were voided, 3 ml fractions (numbers38-80) were collected. NMR was consistent with the desired maleamic acidintermediate.

85 mg of this maleamic acid was dissolved in 4 ml of CHCl₃. 151 μl of1.6 mm acetic anhydride was added followed by 46 mg of anhydrous sodiumacetate and the mixture was stirred overnight at 20°-25°. TLC revealedproduct formation which was worked up by evaporation under reducedpressure to yield crude product. The entire crude was chromatographed ona medium pressure sytem utilizing a 25 g (230-400 mesh) silica gelcolumn, eluted with CH₂ Cl₂ /10% NH₄ OH in MeOH (8:1). The column volumefraction plus fractions numbers 1-8, yielded the title compound. MS(M+H)⁺ : 508.3570 (PLA2)

EXAMPLE 177 1-Propanol,3-[[3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]propyl]amino]

2.9 g of estrone methyl ether was slurried in 55 ml of THF and 80 ml ofMeOH. 11 g of 3Å sieves were then added followed by 2 g of1-propanol-3-[(3-aminopropyl)amino]. The pH was lowered to 6-6.5 withglacial acetic acid, 628 mg of sodium cyanoborohydride was added and themixture was refluxed overnight. A sample was removed from the reactionmixture and evaporated under reduced pressure. Several drops ofconcentrated NH₄ OH were added to the sample and it was again evaporatedunder reduced pressure. CH₂ Cl₂ was added to this sample and it wasfiltered, and evaporated under reduced pressure. TLC analysis (CH₂ Cl₂/10% NH₄ OH in MeOH (4:1)) revealed product formation with some startingmaterial remaining. The entire reaction mixture was filtered through apad of celite. The solids were washed with 200 ml of MeOH/THF (4:3),evaporated under reduced pressure and then about 20 ml of concentratedNH₄ OH was added to the residue (pH 10) and this was again evaporatedunder reduced pressure. The entire crude was chromatographed on 285 g of230-400 mesh silica gel (medium pressure system), eluting with CH₂ Cl₂/10% NH₄ OH in MeOH (4:1). After 600 ml of column volumn was voided, thefollowing 12 ml fractions were taken: fraction numbers 15-30 yielded thefirst title compound; NMR confirmed the structure (PLA2). Fractionnumbers 95-190 yielded the second title compound; NMR confirmed thestructure. MS (M+H)⁺ : 401.3173 (PLA2)

EXAMPLE 178 1H-Pyrrole-2,5-dione,1-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-3,4-dimethyl

Following the general procedure of Example 170, but starting with2,3-dimethylamleic anhydride, the title compound was produced, mp79°-80° C. (PLA2)

EXAMPLE 179 1-Azetidinehexanamie,N-(3-methoxyestra-1,3,5(10)-trien-17-yl) 0.208 g of diamine from Example169, 0.415 g of lithium carbonate and 0.114 g of 1,3-dibromopropane weremixed in 5 ml of CH₃ CN. The mixture was heated in a sand bath to 80° C.overnight. The solution was cooled and maded acidic using 1M HCl. Afterstirring for 10 minutes the solution was made basic with 2M NaOH. Thissolution was extracted with CHCl₃ (2×) and the organic layer was washedwith brine and dried (Na₂ SO₄), filtered and concentrated to provide ayellow oil.

This yellow oil was chromtographed on 35 g of 70-230 silica gel andeluted wth CHCl₃ containing 8% of a MeOH/NH₄ OH (9/1) mixture. Aninitial fraction of 70 ml was collected followed by 3 ml fractions.Fraction numbers 67-90 were nearly homogeneous by TLC and were combinedand concentrated to provide a yellow oil.

This yellow oil was chromatographed on 22 g of 40-60 μm silica gel andeluted with CHCl₃ containing 10% of MeOH/NH₄ OH (9/1) mixture. Aninitial fraction of 75 ml was collected followed by 3 ml fractions.Fractions 3-20 contained the desired intermediate product as a yellowoil.

This yellow oil was chromatographed again on the same column and elutedwith CHCl₃ containing 7.5% of a MeOH/NH₄ OH (9/1) mixture. An initialfraction of 85 ml was collected followed by 3 ml fractions. The productcame off within the first 85 ml and fractions 1-10. Solvent evaporationprovided complete recovery of the material.

The oil was chromatographed a final time on the same column and elutedwith CHCl₃ containing 3% of a MeOH/NH₄ OH (9/1) mixture. An initialfraction of 75 ml was collected followed by 3 ml fractions. Fractions31-80 were homogeneous by TLC and were combined and concentrated toprovide a light yellow oil. This became an off-white solid, m.p. 45°-47°C. NMR was consistent with the desired product. (PLA2)

EXAMPLE 180 Acetamide,2-chloro-N-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]

0.154 g of the diamine from Example 169 was added to 2 ml of CHCl₃. Tothis was added Et₃ N in 1 ml of CHCl₃. The contents were cooled in anacetone-dry ice bath. To this was added 53.4 mg of chloroacetyl chloridein 1.5 ml of CHCl₃. After stirring in the dry ice bath for 15 minutes,the solution was allowed to warm to 20°-25°. Following workup a lightyellow oily soild was produced.

The yellow oily solid was chromatographed on 22 g of 40-60 μm silica geleluting with CHCl₃ containing 3% of a MeOH/NH₄ OH (9/1) mixture. Two 25ml fractions were collected followed by 3 ml fractions. Fractions 71-120were nearly homogeneous by TLC and were combined and concentrated togive a light yellow oil. M.S. results showed that the isolated oil wasthe title compound. MS (M+H)⁺ : 461.2916 (PLA2)

EXAMPLE 181 Butanediamide,N'-[5-[[4-[[5-(acetylhydroxyamino)penyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-hydroxy-N-[5-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]pentyl

1.49 g of estrone methyl ether was slurried in 100 ml of THF and 200 mlof MeOH. 47 g of 3Å sieves were then added followed by 2 g ofdeferoxamine mesylate salt. The pH was then lowered to 6.0-6.5 withglacial acetic acid and 188 mg of sodium cyanoborohydride was added.This was refluxed for about 4 days. TLC (CHCl₃ /25% NH₄ OH in MeOH(4:1)) revealed reaction completion. The material was filtered throughcelite, the solids washed with 200 ml of MeOH, and evaporated underreduced pressure, to yield crude product. The crude product waschromatographed on 1000 g of 70-230 mesh silica gel eluting with CHCl₃/25% NH₄ OH in MeOH (4:1). After an initial fraction of 1300 ml wasvoided, fractions (numbers 1-100) were collected which contained crudeproduct. The crude product was rechromatographed on 100 g of 230-400mesh silica gel (medium pressure system) eluting with CHCl₃ /25% NH₄ OHin MeOH (4:1). After a 100 ml column volume was voided, 6 ml fractionswere collected. Fraction number 8-12 yielded the title compound. MS(M+H)⁺ : 829.5431 (PLA2)

EXAMPLE 182 1H-Pyrrole-2,5-dione,1-[3-[[3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]propyl]amino]propyl]-,didhydrochloride

2.0 g of the free amineN-(3-aminopropyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,3-propanediaminewas dissolved in 9.43 ml of a 0.53M HCl/MeOH solution (5.0 mm) andstirred for 30 min. The MeOH was then blown-off by a stream of nitrogen.20 ml of CHCl₃ was added followed by 2.64 g of 18-crown-6. This mixturewas stirred for 30 min. Then a solution containing 0.7 ml Et₃ N and 2.46g of Boc-ON dissoved in 20 ml of CHCl₃ was added in a dropwise mannerover 20 min. This mixture was stirred at 20°-25° overnight. TLC (CHCl₃/10% NH₄ OH in MeOH (8:1)) revealed product formation. This material wasevaporated under reduced pressure to yield crude product. The entirecrude was chromatographed on 285 g of 230-400 mesh silica gel (mediumpressure), eluting with CHCl₃ /10% NH₄ OH in MeOH (8:1). After 750 ml ofcolumn volume was collected 7 ml fractions (numbers 60-170) were taken.NMR confirmed the intermediate tBOC-steroid maleamic acid was produced.

1.2 g of the tBOC-steroid so produced was dissolved in 100 ml ofo-xylene, 235 mg of maleic anhydride was added and the mixture washeated to reflux. After 1 hour, TLC (CHCl₃ /10% NH₄ OH in MeOH (8:1))revealed product formation. The intermediate maleamic acid was alsopresent and therefore refluxing was continued while adding 100 mg ofmaleic anhydride until some red solid appeared in the reaction flask atwhich point the reaction was stopped. The reaction mixture wasevaporated under a high vacuum to yield crude product. The entire crudewas chromatographed on a 230-400 mesh silica gel medium pressure column,eluting with CHCl₃ /10% NH₄ OH in MeOH (8:1). After 400 ml of columnvolume was voided, 6 ml fractions (numbers 9-30) were taken containingthe tBOC-maleimide as confirmed by NMR.

50 mg of the t-BOC-maleimide was dissolved in a minimal amount of CH₂Cl₂, and then the CH₂ Cl₂ was blown off with a stream of N₂. 3 ml of a2.8M HCl solution in EtoAc was added. After 4 hours, 3 ml more of 2.8MHCl/EtoAc was added and then the mixture was placed in a -78° freezerovernight. The next morning the reaction was stirred for 4 hours at20°-25° , after which TLC (CHCl₃ (10% NH₄ OH in MeOH (8:1)) indicatedonly a trace of starting material remaining. The solid was filtered andplaced on a high vacuum (20°-25°, 0.1 torr) for 2 hours to yield thetitle compound. MS (M+H)⁺ : 480.3221 (PLA2)

EXAMPLE 183 1,3-Propanediamine,N-[3-(3-aminopropoxy)estra-1,3,5(10)-trien-17-yl]N,N',N'-trimethyl

A flame dried 25 ml two necked round bottomed flask, equipped with a 10ml constant pressure addition funnel, magnetic stir bar, and nitrogeninlet tube, was charged with the 0.175 g of NaH dispersion. Thisdispersion was washed three times with hexane; each time the hexane wassuctioned out. The NaH was covered with 4 ml of DMF. To this mixture wasadded 0.401 g of the title compound from Prepareation 11 in 10 ml of DMFover a 25 minute period with stirring in a cold water bath. Stirring wasthen continued at 20°-25° for 1 hour. To this was added 0.332 g ofBoc-3-amino-1-propanol mesylate in 2 ml of DMF over a period of 5minutes. Stirring was continued at 20°-25° for 1 hour. The reactionmixture was poured into ice water, CHCl₃ was added, and the resultinglayers separated. The water layer was washed once with CHCl₃. Thecombined organic layers were washed with brine, filtered, dried (MgSO₄)and concentrated under high vacuum to give a yellow oil.

This oil was chromatographed on 71 g of 70-230 mesh silica gel elutingwith CHCl₃ containing 10% of a MeOH/NH₄ OH (9/1) mixture. Initialfractions of 25 ml (3) were collected followed by 5 ml fractions (actualcolumn volume was approximately 150 ml). Fraction numbers 39-88 werehomogeneous by TLC (CHCl₃ containing 10% of a MeOH/NH₄ OH (9/1) mixture)and were combined and concentrated to give a light yellow oil.

A flame dried 2-necked 25 ml round bottomed flask, equipped with anitrogen inlet tube, rubber stopper and magnetic stir bar, was chargedwith 0.371 g of this light yellow oil and 10 ml of CH₂ Cl₂. To thissolution was added 0.67 ml trifluoroactic acid via syringe. By TLC thereaction appeared nearly complete after 2.5 hours and was worked up.Solvent evaporation gave a yellow oil and a second product.

The combined oils were chromotagraphed on 70 g of 70-230 mesh silica geleluting with CHCl₃ containing 12% of a MeOH/NH₄ OH (9/1) mixture.Initial fractions of 25 ml (6) were colleted followed by 5 ml fractions.Fraction numbers 44-90 were nearly homogeneous by TLC and were combinedand concentrated to provide the title compound as a light yellow oil. MS(M+H)⁺ : 428.3604 (PLA2)

EXMPLE 184 1H-Pyrrole-2,5-dione,1-[3-[[17-[[3-(dimethylamino)propyl]methylamino]estra-1,3,5(10)-trien-3-yl]oxy]propyl]

88.9 mg of the title compound from Example 183 and 34.9 mg of maleicanhydride were added to a 10 ml round bottom flask in 5 ml of o-xylene.The solution was rapidly heated in a sand bath to a temperature of135°-142° C. By TLC after 0.5 hour the reaction was complete. Aftercooling the reaction mixture was worked up. Solvent evaportion underhigh vacuum provided a white solid.

To ensure the product was pure the solid was chromatographed on 22 g of40-60 μm silica gel eluting with CHCl₃ containing 8% of a MeOH/NH₄ OH(9/1) mixture. Two fractions of 25 ml were collected followed by 2.5 mlfractions. Fraction numbers of 19-43 were nearly homogeneous by TLC(CHCl₃ containing 10% of a MeOH/NH₄ OH (9/1) mixture). The fractionswere combined and concentrated to give the title compound as a whitesolid, m.p. 117°-120° C. NMR confirmed the production of this compound.(PLA2)

EXAMPLE 185 L-Lysine, N6-(3-methoxyestra-1,3,5(10)-trien-17-yl)

1.1 g of the 3-methyl estrone ether was slurried in 20 ml of THF and 30ml of MeOH. 4 g of 3Å sieves was then added followed by 1.51 g of 60-tBoc lysine methyl ester. The pH was then lowered with glacial aceticacid to 6.0. Then 243 mg of sodium cyanoborohydride was added and thereaction was refluxed overnight. TLC (CHCl₃ /10% NH4OH in MeOH (20:1))revealed product formation. The reaction was stopped, allowed to cool,filtered through celite, washed with (4:3) MeOH/THF (solids) andevaporated under reduced pressure to yield tBOC steroid. The entirecrude was chromatographed on 285 g of 230-400 mesh silica gel (mediumpressure) column. After 500 ml of column volume was voided, 6 mlfractions (numbers 1-70) were taken. NMR confirmed that the desired tBocsteroid was produced.

50 mg of the tBoc steroid was dissolved in 5 ml of CH₂ Cl₂ and cooled to0° C. 2.46 ml of trifluoroacetic acid was then added and the mixture wasgradually warmed to 20°-25°. After 2 hours TLC (CHCl₃ /10% NH₄ OH inMeOH (8:1)) revealed product formation. The reaction mixture wasevaporated under reduced pressure to yield crude product. The entirecrude was chromatographed on 7 g of 70-230 mesh silical gel, elutingwith CHCl₃ /10% NH₄ OH in MeOH (8:1). After 5 mls was voided, 1 mlfractions (numbers 16-24) were taken which by TLC contained the desiredmethyl ester steroid. NMR confirmed its production.

30 mg of the methyl ester steroid was dissolved in 1 ml of MeOH and then70 μl of 1.0M KOH solution (aqueous) was added. 0.5 ml aliquots of H₂ Owere then added with stirring overnight until the reaction was complete(about 4.0 ml total). TLC (CHCl₃ /10% NH₄ OH in MeOH (8:1)) revealedproduct formation. The reaction mixture was evaporated under reducedpressure and azeotroped with CH₂ Cl₂ (5×) to yield crude product. Theentire crude was dissoved in a small volume of CH₂ Cl₂, and ether wasadded to produce a filterable solid precipitate. Upon filtering thesolid was isolated and was tacky to the touch. The solid was dissolvedin CH₂ Cl₂ and evaporated under pressure, to yield the title compound.NMR confirmed the structure. MS (M+H)⁺ : 453 (PLA2)

EXAMPLE 186 2H-1,4-Diazepin-2-one, hexahydro-4,8-3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]propyl]

23 mg of 2-methylimidizole was dissolved in 2 ml of CH₂ Cl₂ and cooledto -10° C. 15 μl of bromoacety bromide was then added and stirred at-10° C.-0° C. for 1 hour. The reaction mixture was warmed to 20°-25° anda solution containing 30 mg ofN-(3-aminopropyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,3-propanediaminein 2 ml of CH₂ Cl₂ was added dropwise over 30 minutes. This was stirredat 20°-25° for 2 hours. TLC (CHCl₃ /25% NH₄ OH in MeOH (4:1)) revealedproduct formation. The reaction mixture was evaporated under reducedpressure to yield crude product. The entire crude was chromatographed on8 g of 70-230 mesh silica gel, eluting with CH₂ Cl₂ /10% NH₄ OH in MeOH(4:1). After 10 ml of column volume was voided 1 ml fractions werecollected. Preparation A: fractions numbers 25-30.

Preparation A was chromatographed with 7-8 g of 230-400 mesh silica gel,eluting with CH₂ Cl₂ /10% NH₄ OH in MeOH (6:1). After 5 ml of columnvolume was voided, 0.5-1.0 ml fractions (numbers 8-18) were takencontaining the title compound. By I.R., C-13, 200 MH_(z) NMR the titlecompound was produced. MS (M+H)⁺ : 440.3322 (PLA2)

EXAMPLE 187 2-Butenoic acid,4-[[3-[acetyl[3-[(3-methoxyestra-1,3,5(10-trien-17-yl)amino]propyl]amino]propyl]amino]-4-oxo

100 mg ofN-(3-aminopropyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,3-propanediaminewas dissolved in 4 ml of CHCl₃ and a solution containing 27 mg of maleicanhydride in 1 ml of THF was added. After 1 hr TLC analysis (CHCl₃ /25%NH₄ OH in MeOH (4:1)) revealed some starting amine left. Another 10 mgof maleic anhydride in 0.5 ml of THF was added. After 20 minutes TLCanalysis revealed pressure and the entire residue was chromatographed on8 g of 70-230 mesh silica gel, eluting with CHCl₃ /25% NH₄ OH in MeOH(4:1). After 10 ml of column volume was voided, 0.5-1.0 ml fractions(numbers 16-35) were taken which contained the maleamic acidintermediate as confirmed by NMR.

48 mg of this maleamic acid was dissolved in 2 ml of CHCl₃, and 16 μl ofglacial acetic acid was added and stirred for 15 minutes. 39 μl ofacetic anhydride was then added followed by 3-5 mg of NaO₂ C₂ H₃. Thismixture was stirred at 20°-25° for 30 minutes. TLC analysis (CHCl₃ /25%NH₄ OH in MeOH (4:1)) revealed product formation. The entire reactionmixture was evaporatead under reduced pressure and the white residue waschromatographed on 8 g of 70-230 mesh silica gel, eluting with CHCl₃/25% NH₄ OH in MeOH (4:1). After 10 ml of column volume was voided,0.5-1.0 ml fractions (numbers 9-14) were taken containing the titlecompound, mp 121°-23° C. (PLA2)

EXAMPLE 188 L-Lysine, N6-[1-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-2,5-dioxo-3-pyrrolidinyl]-methyl ester

Lysine methyl ester didhydrochloride, 2 ml of ethanol, and 0.110 ml Et₃N were added together and stirred until the lysine was completelydissolved. This entire solution was added to a heated solution (37°-39°C.) of the title compound from Example 170 and 30 ml of absolute ethanoland stirred for 4 hours. By TLC the reaction was complete.

Water was added to the solution and the pH was raised to 11 using diluteK₂ CO₃ (2%). The solution was extracted with CHCl₃ (2×) and the organicphase washed with brine, dried (MgSO₄), filtered, and concentrated toprovide an oily white solid.

The oily white solid was chromatographed on 22 g of 40-60 μm silica geleluting with CHCl₃ containing 5% of a MeOH/NH₄ OH (9/1) mixture. Two 25ml fractions were collected followed by 3 ml fractions. Fraction numbers69-93 contained the desired product along with impurities. Solventevaporation provided 0.156 g of a yellow oil. This oil wasrechromatographed on 22 g of 40-60 μm silica gel using the same solventsystem. After two 25 ml fractions were voided, 3 ml fractions werecollected. Fraction numbers 64-83 were collected and solvent evaporationprovided the title compound as a light yellow oil product. MS (M+H)⁺ :625.4346 (PLA2)

EXAMPLE 189 2,5-Pyrrolidinedione,3-methoxy-1-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl];Butanoic acid,3-methoxy-4[[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]amino]-4-oxo, methyl ester; Butanoic acid,2-methoxy-4-[[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]amino]-4-oxo-,methyl ester

The title compound from Example 170 was heated in MeOH in a sand bath toa temperatuare of 55° C. After 2 hours the reaction was complete by TLC.The solution was allowed to cool and the MeOH evaporated under reducedpressure to provide a light yellow oil. While in the refrigerator theoil became an off-white semi-solid.

The semi-solid was chromatographed on 22 g of 40-60 μm silica geleluting with CHCl₃ containing 4% of a MeOH/NH₄ OH (9/1) mixture.Initially, two-25 ml fractions were collected followed by 2.5 mlfractions. Fraction numbers 18-39 were homogeneous by TLC andcorresponded to the first title compound (10A) (PLA2). Fraction numbers45-90 were homogeneous by TLC and corresponded to the second titlecompound (10B). Fraction numbers 103-123 were homogeneous by TLC andcorresponded to the third title compound (10C). The appropriatefractions were combined and concentrated to give an oil for 10A, an oilfor 10B, and an oil for 10C. MS (M+H)⁺ : 10A=497.3390, 10B=529.3669,10C=529.3613 (PLA2)

EXAMPLE 190N-(5-fluoro-2,4-dinitrophenyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,6-hexanediamine

0.151 g of the title compound from Example 169 was dissolved in 20 ml ofMeOH. To this was added 0.6 ml Et₂ N, followed by 0.312 g1,5-difluoro-2,4-dinitrobenzene in 10 ml of MeOH. By TLC after 15minutes the reaction was complete. The solution was diluted with CHCl₃and washed with H₂ O. The H₂ O layer was extracted with additional CHCl₃and the combined organic layers were washed with brine and dried (Na₂SO₄), filtered, and concentrated to give an oil.

The oil was chromatographed on 50 g of 70-230 mesh silica gel elutingwith CHCl₃ containing 3.5% of a MeOH/NH₄ OH (9/1) mixture. An initialfraction of 50 ml was collected followed by 5 ml fractions. Fractions17-45 were homogeneous by TLC and were combined and concentrated to givea semi-solid. NMR was consistent with the desired product. MS (M+H)⁺ :569.3146

EXAMPLE 191N-(2,4-dinitrophenyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,6-hexanediamine

Following the general procedure of Example 190 but starting with2,4-dinitrofluorobenzene the title compound is produced, mp 90°-92° C.

EXAMPLE 192N-[4-(4'-Cyclohexylcyclohexylidene)cyclohexyl]-N-(3-pyridinyl)methylaminesand

To a solution of 3-aminomethylpyridine (0.800 ml) and acetic acid (0.65ml) in methanol (16 ml) was added the title compound from Preparation 19(1.0 g) and solid sodium cyanoborohydride (0.252 g of 95% quality) in aN₂ atmosphere. A vigorous evolution of gas occurred when the hydride wasadded. TLC after three hours indicated that the reaction was complete.The reaction mixture was poured into aqueous 1N NaOH and the pH of themixture was adjusted to pH>10. The aqueous mixture was extracted withether (4X), and the pooled ether extracts were washed with water, dried(Na₂ SO₄), filtered, and concentrated to give a white solid.Chromatography (190 g of 40-63 μm silica gel, 96.3-3-0.2 CHCl₃-methanol-ammonium hydroxide, 40 ml fractions) of this solid gave a lesspolar isomer eluting in fractions 14-18 and a more polar isomer elutingin fractions 20-30.

The less polar isomer was recrystallized from ether-pentane, givingcolorless crystals, mp 93.5°-94.5° C.

EXAMPLE 193 N-Benzylestra-1,3,5(10)-trien-17β-amine

To a solution of benzylamine (11.2 ml) in dry methanol (75 ml) was addedsequentially acetic acid (6.16 ml), estra-1,3,5(10)-trien-17-one (5.21g) sodium cyanoborohydride (1.35 g), and THF (60 ml) under a N₂atmosphere. The resulting mixture was stirred overnight at roomtemperature, concentrated in vacuo, diluted with water (200 ml), andmade alkaline with aqueous sodium hydroxide (50 ml). The suspension wasextracted with ethyl acetate (3×200 ml) and the combined extracts weredried over MgSO₄, filtered and concentrated to an oil. This product waschromatographed (300 g silica gel 0.4-4-4-96 Et₃ N-acetone-hexane, 20 mlfractions) and gave impure product in fractions 53-79 and pure productin fractions 80-135. The latter were combined and after removal of thesolvent gave the title compound as crystals, mp 71°-74° C. (PLA2,diabetes)

EXAMPLE 1943-Methoxy-N-[2-(4'-pyridinyl)ethyl]estra-1,3,5(10)-trien-17β-amine

To methanol (110 ml) were added sequentially 4-(2-aminoethyl)pyridine(20.4 g, Reilly), acetic acid (20 ml, 20 g), estrone methylether (9.5g), sodium cyanoborohydride (2.09 g), and THF (80 ml). The resultingmixture was stirred at room temperature for 30 hours. The now clearsolution was concentrated under reduced pressure to a yellow solidresidue. Water (300 ml) and aqueous 50% sodium hydroxide were added tothe residue and the resulting mixture was extracted 2X with ethylacetate. The extracts were dried (MgSO₄), filtered, and concentrated. Asolid yellow residue was obtained. Recrystallization of the solid fromacetone gave first crop of crystals, mp 143°-147° C., and a second cropof crystals. Recrystallization of 1.00 g from acetone gave 0.724 g ofthe title compound as nearly colorless crystals, mp 145°-147° C. (PLA2-,diabetes)

EXAMPLE 1953-Methoxy-N-[4-(3'-pyridinylmethyl)phenyl]estra-1,3,5(10)-trien-17β-aminedihydrochloride

A solution of estrone methyl ether (8.52 g) and3-(4'-amino)benzylpyridine (5.52 g) in toluene (100 ml) was heated toreflux temperature. Water in the condensate was collected in aDean-Stark trap. After 23 hours the solution was concentrated underreduced pressure and methanol (70 ml) and tetrahydrofuran (40 ml) wereadded to the residue. The resulting solution was stirred at roomtemperature, sodium cyanoborohydride (3.5 g) and acetic acid (3.6 ml,3.6 g) were added, and stirring was continued overnight. TLC (10% of 10%NH₄ OH in CH₃ OH in CHCl₃) showed starting ketone and two more polarcomponents. Stirring another 4 hours with additional sodiumcyanoborohydride did not change the TLC picture significantly. Water wasadded to the reaction solution and the solution was concentrated underreduced pressure, giving a solid residue. Water (200 ml) and 50% aqueousNaOH were added to the residue. The residue was broken up with a spatulaand the mixture was extracted with ether (2X), the ether extracts weredried (MgSO₄), filtered and concentrated. A gummy, yellow residue wasobtained and was chromatographed (395 g silica gel, 30% ethyl acetate inhexane, 45 ml fractions) using low pressure liquid chromatography. Thedesired product was eluted in fractions 80-122.

A part of the product (4.580 g) was dissolved in ether and ethereal NClwas added. The precipitate was yellow at first but with more HNl, theprecipitate became white. The ether was blown off with a stream of N₂.The residual solid was crystallized from methanolether and gave thetitle compound, mp 170°-215° C. (PLA2-, diabetes)

EXAMPLE 196 N-Diphenylmethyl-3-methoxyestra-1,3,5(10)-trien-17β-amine

A solution of aminodiphenylmethane (5.50 g) in methanol (100 ml) wasstirred at room temperature. Added sequentially to this solution wereglacial acetic acid (1.8 ml, 1.8 g), estrone methyl ether (8.52 g),sodium cyanoborohydride (2.3 g), and THF (80 ml). The mixture wasstirred at room temperature for 8 days. Additional NaB(CN)H₃ (1.5 g),amine (5.5 g), and acetic acid (1.8 g) were added after four days of thereaction time. Excess solvent was removed under reduced pressure andwater (300 ml) was added to the solid residue. The mixture was madealkaline with 50% aqueous NaOH and was extracted with ether (3X). Theextracts were dried (MgSO₄), filtered, and concentrated, giving an oilyresidue. This crude product was chromatographed (500 g silica gel, 5%ethyl acetate-hexane, 350 ml fractions) on a gravity column. The desiredproduct was eluted in fractions 4-7, and after recrystallization frommethanol gave a first crop of crystals, mp 127°- 129° C., and a secondcrop. A sample was recrystallized again from methanol and gave ananalytical sample of the title compound, mp 127°-129° C. (PLA2,diabetes)

EXAMPLE 197 N-[2-(2'-Pyridinyl)ethyl]estra-1,3,5(10)-trien-17β-amine

To a solution of 2-(2-amino)ethylpyridine (12.91 ml, 13.2 g) in methanol(75 ml) were added sequentially, under N₂, acetic acid (12.31 ml),estrone methyl ether (5.21 g) sodium cyanoborohydride (1.35 g), andtetrahydrofuran (60 ml). The resulting mixture was stirred at roomtemperature for 40 hours, then concentrated in vacuo, diluted with water(200 ml), and aqueous 50% sodium hydroxide (50 ml). This mixture wasextracted with ethyl acetate (3×200 ml) and the combined extracts weredried (MgSO₄), filtered and concentrated to a solid. The crude productwas chromatographed (300 g silica gel, 0.5% triethylamine/10%acetone/hexane, 30 ml fractions), giving the desired product infractions 61-142. These fractions were pooled and crystallized from hothexane to give a first crop of title compound, mp 74°-76° C., and asecond crop of title compound. (PLA2)

EXAMPLE 198 N-(3'-Pyridinyl)methylestra-1,3,5(10)-17β-amine

A solution of 3-aminomethylpyridine (10.65 ml) in methanol (75 ml) underN₂ was treated sequentially with acetic acid (12.3 ml), estrone methylether (5.21 g), sodium cyanoborohydride (1.35 g), and THF (60 ml). Themixture was stirred at room temperature for 24 hours, then concentratedin vacuo, diluted with water (200 ml) and 50% aqueous NaOH, andextracted with ethyl acetate (3×200 ml). The extracts were combined,dried (MgSO₄), filtered, and concentrated to a white solid. The crudeproduct was chromatographed on silica gel (320 g) in 0.4%triethylamine/8% acetone/hexane while collecting fractions of 40 mlvolume. The desired product was eluted in fractions 163-270, which werecombined and crystallized from hot hexane to give the title compound, mp92°-93° C. (PLA2, diabetes)

EXAMPLE 199N-[2-(2'-Phenyl)ethyl]-3-methoxy-estra-1,3,5(10)-trien-17β-amine

Phenethylamine (13.33 ml) was dissolved in anhydrous methanol (75 ml)and treated under nitrogen with stirring with glacial acetic acid (6.31ml), followed by estrone methyl ether (5.97 g), sodium cyanoborohydride(1.38 g, 21 mmoles) and freshly distilled THF (60 ml). The resultingsuspension was stirred at 60±5° under nitrogen for 20 hours, cooled toroom temperature, concentrated in vacuo and diluted with water (200 ml)then treated with 50% aqueous sodium hydroxide (50 ml). The resultingsuspension was extracted with ethyl acetate (3×200 ml) and the combinedextracts were dried over magnesium sulfate, filtered and concentrated toa semi-solid.

The crude product was chromatographed on silica gel (300 g) in 0.75%triethylamine/17.5% acetone/hexane while collecting 40 ml fractions.Fraction numbers 18-21 were concentrated to give a solid; fractionnumbers 22-34 were combined and concentrated to give a solid which wasrecrystallized from hot acetone hexane to give the crystalline titlecompound, mp 137°-9° C. (PLA2-, diabetes)

EXAMPLE 200 (1'S,2'S)-N-(1',3'-Dihydroxy-1'-phenyl)isopropyl-3-methoxyestra-1,3,5(10)-triene-17β-amine

1S,2S-(+)-2-Amino-1-phenyl-1,3-propanediol (22.26 g) glacial acetic acid(7.61 g), estrone methyl ether (7.20 g), tetrahydrofuran (65 ml) andsodium cyanoborohydride (1.67 g) were added to methanol (88 ml). Theresulting mixture was stirred at room temperature for 22 hours. TLC (40%ethyl acetate-hexane) after 44 hours showed remaining starting materialand after three days the reaction was completed. Excess solvent wasremoved under reduced pressure. Water was added and made basic (pH 9)with aqueous 50% NaOH. The cloudy mixture was extracted with ether (3X).The combined extracts were dried (NaSO₄), filtered and concentrated to awhite solid. TLC showed a polar impurity. The solid was mixed with 33 gof flash silica gel and ethyl acetate. The solvent was removed underreduced pressure and the silica gel was poured on top of thechromatographic column. The compound was eluted with 50% ethylacetate-hexane. The solid was recrystallized from ethyl acetate to givea first crop of colorless crystals, mp 194°-195° C. A portion of thefirst crop ws recrystallized, mp 192°-193° C. A second crop wasobtained, mp 192°-193° C. (PLA2-, diabetes)

EXAMPLE 201N-(2'-(4"-Hydroxyphenyl)ethyl)-3-methoxyestra-1,3,5-(10)-triene-17β-amine

Tyramine (5.27 g), glacial acetic acid (5.27 g), estrone methyl ether(4.92 g) sodium cyanoborohydride (1.09 g), tetrahydrofuran (43 ml) wereadded to methanol (58 ml). The reacting mixture was allowed to stir for21 hours at room temperature. TLC (ethyl acetate) revealed theinformation of a more polar product (Rf 0.25). After 19 hours TLCrevealed that the starting material still remained in the reactionsolution. The system was warmed at 40 C. in an oil bath. The solutionremained as a white undissolved solid. After 29 hours the systsem wasallowed to reach room temperature. Excess solvent was removed underreduced pressure. Water was added and made basic (pH 9) with aqueous 50%NaOH. The white solid did not dissolve in ether and was slightly solublein ethyl acetate. The solid was filtered and mixed with 34 g of flashsilica gel and ethyl acetate. The solvent was removed under reducedpressure. The silica gel was poured on top of the chromatographiccolumn. The compound was eluted with ethyl acetate. Solid was obtainedand was recrystallized from ethyl acetate, mp 184°-185° C. (diabetes)

EXAMPLE 202 N-Benzyl-5α-androstan-17β-amine

5α-Androstan-17β-amine (10.946 g), glacial acetic acid (2.39 g), sodiumcyanoborohydride (0.50 g), benzaldehyde (0.84 g) and tetrahydrofuran (50ml) were added to methanol (25 ml). The resulting mixture was stirred atroom temperature for 48 hours. Excess solvent was removed under reducedpressure. Water was added and made basic (pH 9) with 50% aqueous NMaOH.The mixture was extracted with ether (3X). The combined extracts weredried, filtered and concentrated to a solid. The solid was mixed with 20g of flash silica gel and dissolved in ethyl acetate. The solvent wasremoved at reduced pressure and the silica gel was poured on top of thechromatographic column. The compound was eluted with 5%triethylamine-ethylacetate. 6 fractions (200 ml) were collected. An oilwas obtained and was dissolved in hot methanol. The solution was cooledgiving colorless crystals, mp 85.5°-87.5° C. (PLA2, diabetes)

EXAMPLE 203 N-(3-Dimethylaminopropyl)-N-[2-(2'-fluoro-1',1"-biphen-4'-yl)ethyl]amine

A solution of the title compound from Example 55 (3.28 g) in dioxane (75ml) was added under N₂ to a slurry of LiAlH₄ (0.759 g) in dioxane. Theresulting mixture was stirred at room temperature for 20 hours afterwhich TLC of a quenched aliquot revealed only starting material. Themixture was heated at 60° C. for 24 hours, additional LiAlH₄ (1.14 g)was added, and the mixture was heated at 60° C. for an additional 24hours. The reaction was cooled in an ice bath and quenched by thecareful addition of, first, ethyl acetate and then water. The mixturewas filtered through celite and the filtrate layers were separated. Theaqueous layer was further extracted with ethyl acetate, the combinedextracts were dried (Na₂ SO₄), filtered, and concentrated. The residuewas chromatographed over silica gel (40-63 μm, 395 g) usingchloroform-methanol-ammonium hydroxide (90-9-1; 50 ml fractions) as thesolvent system. The title compound wsa eluted in fractions 31-42 whichcontained 0.626 g of oil: IR (liquid film) 2958, 2940, 2894, 2874, 1625,1484, 1461, 1450, 1418, 1269, 1132, 1041, 836, 767, 725, 698 cm⁻.(PLA2-, diabetes)

EXAMPLE 204 N-((4'-Chloro)benzyl)-5α-androstan-17β-amine

4-Chlorobenzaldehyde (0.464 g), 5α-androstan-17β-amine (4.40 g), glacialacetic acid (0.99 g), sodium cyanoborohydride (0.21 g) andtetrahydrofuran (5 ml) were added to methanol (11 ml). The resultingmixture was warmed to dissolve the solid in solution and allowed to stirovernight at room temperature. Excess solvent was removed under reducedpressure. Water was added and made basic (pH 9) with 50% aqueous NaOH.The mixture was extracted with ether and the organic layer becamecloudy. Celite was used to filter the organic layer. The aqueous layerwas washed with ether and the combined organic layers were dried(NaSO₄), filtered and concentrated. The crude solid was mixed with 10 gof flash silica gel and dissolved in ethyl acetate. The solvent wasremoved and the silica was poured on top of the chromatographic column.The compound was eluted with 50% ethyl acetate-hexane. The titlecompound was obtained as a solid which was recrystallized from acetone(0.364 g) mp 101°-102° C. (PLA2 , diabetes)

EXAMPLE 205N-(4'-Hydroxy-3'-methoxybenzyl)-3-methoxyestra-1,3,5(10)-triene-17β-amine

4-Hydroxy-3-methoxybenzylhydrochloride amine (8.32 g), estrone methylether (2.51 g), sodium cyanoborohydride (0.55 g) were added to methanol(30 ml) followed by tetrahydrofuran (22 ml). The solution was allowed tostir for hour 4 days at room temperature. TLC (ethyl acetate) revealedthe formation of a more polar compound and the presence of some startingmaterial. The solution was heated to reflux for a few more days. TLCrevealed the reaction did not complete. Excess solvent was removed atreduced pressure. Water was added and made basic (pH 9) with aqueous 50%NaOH. The solution was extracted with ether (3X). The combined extractswere dried (Na₂ SO₄), filtered and concentrated. The oil was mixed with20 g of flash silica gel and ethyl acetate. The solvent was removedunder reduced pressure and the silica gel was poured on top of thechromatographic column. The compound was eluted with ethyl acetate. Thetitle compound was obtained as a solid and was recrystallized from ethylacetate (0.488 g), mp 169°-169.5° C. (PLA2, diabetes-)

EXAMPLE 206 N-[(5α,17β)-Androstan-17-yl]-1,6-hexanediamine

A 100 ml round-bottomed flask, equipped with a magnetic stir bar,condenser, and nitrogen inlet tube, was charged with 1,6-hexanediamine(0.904 g) and 15 ml of methanol. The solution was treated with glacialacetic acid until the pH was 6.7. To this mixture was added 15 ml oftetrahydrofuran, 5α-androstan-17-one (0.704 g) and sodiumcyanoborohydride (0.228 g). The mixture was heated to reflux for 18 h.After cooling, the solution was made basic with aqueous 2M sodiumhydroxide to a pH of 13-14. The solution was extracted with chloroform(3X) and the combined organic layers were washed with water and brine,dried (Na₂ SO₄), filtered, and concentrated to give 1.2 g of an oil.

The oil was chromatographed on 87 g of 230-400 mesh silica gel, elutingwith chloroform containing 20% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 150 ml was collected followed by 5 mlfractions. Fractions 12-20 were impure by TLC. These fractions werecombined, concentrated, and rechromatographed on the same column elutingwith chloroform containing 15% of a methanol/ammonium hydroxide (9/1)mixture. Fractions 18-43 were homogeneous by TLC and were combined withfractions 21-50 which were pure from the first chromatography to give0.656 g of an oil. IR, νmax (neat) 2924, 2853, 1470, 1464, 1447, 1158,827, 797, and 767 cm⁻¹.

EXAMPLE 2071-[6-[[(5α,17β)-Androstan-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione

The title compound from Example 206 (0.393 g) and maleic anhydride(0.180 g) were covered with 12 ml of o-xylene and placed under nitrogen.The mixture was heated with stirring in a sand bath to a temperature of145°-150° C. for 2 hours. After the mixture had cooled, the solvent wasevaporated under high vacuum to give 0.966 g of a semi-solid.

The semi-solid was chromatographed on 50 g of 230-400 mesh silica geleluting with chloroform containing 5% of a methanol/ammonium hydroxide(9/1) mixture. An initial fraction of 125 ml was collected followed by 5ml fracations. Fractions 2-40 contained the desired product along withsmall amounts of lower Rf impurities. The fractions were combined andconcentrated to give 0.194 g of a white solid (m.p. >250° C.). IR νmax(mull) 1706, 1451, 1409, 1392, 843, 833, and 696 cm⁻¹.

EXAMPLE 2083-(3-t-Butoxycarbonyl-aminopropoxy)estra-1,3,5(10)-trien-17-one

A flame dried 1000 ml 3-necked round-bottom flask, equipped with anitrogen inlet tube, magnetic stir bar, 500 ml constant pressureaddition funnel and 125 ml constant pressure addition funnel, wascharged with a 60% sodium hydride dispersion (4.80 g). The dispersionwas washed with hexane (3X) with the solvent removed by suction. Thesodium hydride was covered with 100 ml of dimethylformamide and theestrone (11.1 g) in 250 ml of dimethylformamide was added over 35minutes. The mixture was stirred at room temperature for 1 hour. Themesylate (13.06 g) in 80 ml of dimethylformamide was added to the abovesolution over 10 minutes and the mixture stirred an additional 1.5hours.

The reaction mixture was poured slowly and carefully into ice water. Theaqeueous solution was extracted with hexane/ethyl acetate (1/1) and thenchloroform. The organic phase was washed with brine, dried (MgSO₄),filtered, and concentrated to give 18.4 g of an oil. The oil waschromoatographed on 1600 g of 230-400 mesh silica gel eluting withchloroform/acetone (99/1). An initial fraction of 300 ml was collectedfollowed by 45 ml fractions. Fractions 88-97 and 118-140 containedimpure product as determined by TLC. These fractions were combined,concentrated, and rechromatographed on 300 g of 230-400 mesh silica geleluting with the same solvent. After collecting a 450 ml fraction, 8 mlfractions were collected. Fractions 71-126 were homogeneous by TLC andwere combined with fractions 98-117 from the first chromatography togive 6.15 g of a powder (m.p. 134°-136° C.).

EXAMPLE 209 N-[(17β)-3-[3-t-Butoxycarbonyl-aminopropoxy]estra-1,3,5(10)-trien-17-yl]-1,3-propanediamine

A 25 ml round-bottomed flask, equipped with a magnetic stir bar,condenser, and nitrogen inlet tube, was charged with 1,3-propanediamine(0.31 ml, d=0.888) and 7.5 ml of methanol. The solution was treated withglacial acetic acid until the pH was 6.6. To this mixture was added 7.5ml of tetrahydrofuran, the title compound from Example 208 (0.518 g),and sodium cyanoborhydride (97.1 mg). The mixture was heated to refluxfor 51/2 hours. After cooling the solution was made basic with aqeuos 2Msodium hydroxide to a pH of 13-14. The solution was extracted withchloroform (3X) and the organic phase washed with water and brine, dried(Na₂ SO₄), filtered, and concentrated to give 0.836 g of a colorlessoil.

The oil was chromatographed on 85 g of 230-400 mesh silica gel elutingwith chloroform containing 10% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 130 ml was collected followed by 5 mlfractions. Fractions 86-100 were impure by TLC. These fractions werecombined, concentrated, and rechromatographed on the same column elutingwith chloroform containing 12-15% of a melthanol/ammonium hydroxide(9/1) mixture. Using the same fraction size, fractions 21-40 werehomogeneous by TLC and were combined with fractions 101-134 from thefirst chromatography to give, after trituration with hexane, 0.387 g ofa solid (m.p. 87.5°-89.5° C.).

EXAMPLE 210 1,3-Propanediamine,N-[3-(3-aminopropoxy)estra-1,3,5(10)-trien-17-yl]-, trihydrochloride

A flame dried 25 ml 2- necked round-bottomed flask equipped with anitrogen inlet tube, rubber spetum, and magnetic stir bar, was chargedwith the title compound from Example 209 (0.123 g) and 10 ml of ethylacetate. To this was added 0.3 ml of methanol to completely dissolve thestarting material. A 2.8M solution of HCl in ethyl acetate was added viasyringe (0.4 ml). Over a period of 41/2 hours additional HCl-ethylacetate (1.5 ml) was added. By TLC after 41/2 hours the reaction wascomplete. The addition of ether to ensure precipitation formed a finelydivided solid which was difficult to filter. Therefore the solvent wasevaporated under reduced pressure and the resulting solid trituratedwith hexane. The collected solid was dried under high vacuum overnightto provide 117 mg of a white solid (m.p. 227°-281° C.). (PLA2)

EXAMPLE 211 [U-75733F] 1,3-Propanediamine,N-[3-(3-aminopropoxy)estra-1,3,5(10)-trien-17-yl] -,tris(trifluoroacetate)

A flame dried 25 ml 2-necked round-bottomed flask equipped with anitrogen inlet tube, magnetic stir bar, and rubber septum, was chargedwith the title compound from Example 209 (0.102 g), 5 ml of methylenechloride, and trifluoroacetic acid (0.25 ml). By TLC after 2 hours thereaction was complete with only a minor impurity. Addition of etherprecipitated out the salt which was collected using a fine fritscintered glass funnel. The solid was dried under high vacuum overnightto provide 140 mg of a powder (m.p. 2.3°-207° C.). (PLA2)

EXAMPLE 212N-[(17β)-3-[3-t-Butoxycarbonyl-aminopropoxy]estra-1,3,5(10)-trien-17-yl]-1,6-hexanediamine

A 50 ml round-bottom flask, equipped with a magnetic stir bar,condenser, and nitrogen inlet tube, was charged with 1,6-hexanediamine(0.437 g) and 10 ml of methanol. The solution was treated with glacialacetic acid until the pH was 6.6. To this mixture was added 10 ml oftetrahydrofuran, the title compound from Example 208 (0.509 g), andsodium cyanoborohydride (0.11 g). The mixture was heated to reflux for 5hours. After cooling the solution was made basic with aqueous 2M sodiumhydroxide to a pH of 13-14. The solution was extracted with chloroformand the organic phase washed with water and brine, dried (Na₂ SO₄),filtered, and concentrated to give 0.962 g of an oil.

The oil was chromatographed on 85 g of 230-400 mesh silica gel elutingwith chloroform containing 15% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 130 ml was collected followed by 5 mlfractions. Fractions 34-46 were impure by TLC. These fractions werecombined, concentrated, and rechromatographed on the same column elutingwith chloroform containing 10% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 130 ml was collected followed by 5 mlfractions. Fractions 27-63 were homogeneous by TLC and were combinedwith fractions 47-60 from the first chromatography to give 0.492 g of anoil. In another trial trituration with hexane gave a white solid (m.p.82°-84° C.).

EXAMPLE 213 1,6-Hexanediamine,N-[3-(3-aminopropoxy)estra-1,3,5-(10)-trien-17-yl]-, trihydrochloride

A flame dried 25 ml 2-necked round-bottomed flask equipped with anitrogen inlet tube, rubber septum, and magnetic stir bar, was chargedwith the title compound from Example 212, (0.118 g) and 10 ml of ethylacetate. To this was added 0.3 ml of methanol to completely dissolve thestarting material. A 2.8M solution of HCl in ethyl acetate was added viasyringe (1.3 ml) followed by an additional 1.3 ml of the HCl-ethylacetate solution over a 3 hour period. After a total reaction time of61/2 hours ether was added to ensure complete precipitation of the salt.The salt was collected using a fine frit sintered glass funnel and driedunder high vacuum overnight to provide 106 mg of a solid (m.p. 265°-270°C.). (PLA2)

EXAMPLE 2141-[6-[[(17β)-3-[3-t-Butoxycarbonyl-aminopropoxy]estra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrol-2,5-dione

The title compound from Example 212 (0.301 g) and maleic anhydride (94.7mg) were covered with 15 ml of o-xylene and placed under nitrogen. Thesolution was refluxed for 4 hours. After cooling, the solvent wasevaporated under high vacuum to give 0.67 g of an oil.

The oil was chromatographed on 70 g of 70-230 mesh silica gel elutingwith chloroform containing 5% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 100 ml was collected followed by 5 mlfractions. Fractions 10-30 were homogeneous by TLC and were combined andconcentrated to give, after trituration with hexane, 0.162 g of a yellowsolid (m.p. 103°-105° C.).

EXAMPLE 215 Acetic acid, trifluoro-, compd. with1-[6-[[3-(3-aminopropoxy)estra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione(2:1)

A flame dried 25 ml round-bottomed flask, equipped with a nitrogen inlettube and magnetic stir bar, was charged with the title compound fromExample 214 (0.136 g) and 6 ml of methylene chloride. To this solutionwas added trifluoroacetic acid (0.21 ml, d=1.48). After stirring at roomtemperature for almost 2 hours additional trifluoroacetic acid (0.1 ml)was added to complete the reaction. After a total reaction time of 3hours, ether was slowly added to precipitate the salt. The salt oiledout. The solvent was evaporated under reduced pressure. After repeatedattempts at forming a solid by trituration with ether a light yellowfoam was obtained. This salt was placed under high vacuum for 24 hoursto give 0.160 g of solid. IR, νmax (mull) 1707, 1672, 1611, 1501, 1411,1367, 1203, 1179, 1134, 832, 799, and 696 cm⁻¹. (PLA2)

EXAMPLE 216 N-t-Butoxycarbonyl-6-amino-1-hexanol, methanesulfonate

t-Butoxycarbonyl formation: 6-Amino-1-hexanol (3.13 g) was dissolved in150 ml of tetrahydrofuran. To this was added di-t-butyldicarbonate (5.87g) in 20 ml of tetrahydrofuran over several minutes. The solution wasstirred at room temperature for 50 minutes. The solvent was evaporatedunder reduced pressure and the resulting oil taken up in ethyl acetate.The organic solution was washed with aqueous 5% sodium hydroxide andbrine, dried (MgSO₄), filtered, and concentrated to give 5.77 g of anoil. The product was pure by TLC. (RF=0.55 in ethyl acetate/hexane[7/3]).

Mesylate formation: The entire oil from the previous reaction was takenup in 100 ml of methylene chloride and placed under nitrogen. Thesolution was cooled in a carbon tetrachloride - dry ice bath (-20° C.).Addition of triethylamine (3.91 g) in one portion was followed byaddition of methanesulfonyl chloride (2.4 ml, d=1.48) over severalminutes. By TLC after 35 minutes the reaction was complete. The reactionwas quenched with ice water and the layers separated. The organic layerwas washed with dilute aqueous sodium bicarbonate and brine, dried (Na₂SO₄), filtered, and concentrated to give 7.69 g of a solid (m.p.48°-49.5° C.).

EXAMPLE 217N-[(17β)-3-[6-t-Butoxycarbonyl-aminohexoxy]estra-1,3,5(10)-trien-17-yl]-N,N',N'-trimethyl-1,3-propanediamine

A flame dried 25 ml 2-necked round-bottomed flask, equipped with anitrogen inlet tube, 10 ml constant pressure addition funnel, andmagentic stir bar, was charged with a 60% dispersion of sodium hydride(58 mg). The dispersion was washed with hexane (3X), the hexane beingremoved by suction through a gas dispersion tube. The sodium hydride wascovered with 3 ml of dimethylformamide. Addition of the title compoundfrom Preparation 11 (0.202 g) in 8 ml of dimethylformamide was carriedout over 15 minutes. After stirring for 1 hour at room temperature theboc-mesylate (0.196 g) in 2 ml of dimethylformamide was added. By TLCafter 11/2 hours the reaction was only 20% complete. The solution washeated in an oil bath to a temperature of 55° C. overnight. Aftercooling the reaction was poured into ice water and the aqueous solutionwas extracted with chloroform (3X). The combined organic layers werewashed with brine, dried (MgSO₄), filtered, and concentrated under highvacuum to give 0.401 g of an oil.

The oil was chromatographed on 59 g of 230-400 mesh silica gel elutingwith chloroform containing 7% of a methanol/ammonium hydroxide (9/1)mixture. An initial fraction of 125 ml was collected followed by 5 mlfractions. Fractions 76-92 were impure by TLC and were rechromatographedon 22 g of 230-400 mesh silica gel eluting with chloroform containing 6%of a methanol/ammonium hydroxide (9/1) mixture. An initial fraction of50 ml was collected followed by 5 ml fractions. Fractions 14-21 werehomogeneous by TLC and were combined with fractions 66-75 from the firstchromatography to give 106 mg of a colorless oil. NMR (CDCl₃ ; TMS): δ7.3-7.15, 6.8-6.6, 4.8-4.5, 4.05-3.85, 2.25, 1.45, 0.8

EXAMPLE 218 Acetic acid, trifluoro- , compd. withN-[3-[(6-aminohexyl)oxy]estra-1,3,5(10)-trien-17-yl]-N,N',N'-trimethyl-1,3-propanediamine(3:1)

A flame dried 25 ml round-bottomed flask, equipped with a nitrogen inlettube and magnetic stir bar, was charged with the title compound fromExample 217 (0.106 g) and 6 ml of methylene chloride. To this solutionwas added trifluoroacetic acid (0.25 ml, d=1.48). After 2 hours thereaction was 80% complete by TLC. However, after 5 hours at roomtemperature there was still 5-10% unreacted starting material.Additional trifluoroacetic acid (0.15 ml) was added and stirringcontinued for 45 minutes. To precipitate out the salt, ether was added,but the salt oiled out. Solvent evaporation and trituration with hexaneand ether still provided an oil. By TLC there remained ˜5% startingmaterial. The oil was again taken up in methylene chloride andtrifluoroacetic acid (0.2 ml) was added. After 2 hours at roomtemperature there remained ˜5% of starting material. Solvent evaporationand concentration provided 0.184 g of an oil. IR: νmax (neat) 3035,2941, 2869, 1779, 1744, 1676, 1612, 1501, 1477, 1428, 1203, 1185, 1140,837, 799, 722, and 707 cm⁻¹. (PLA2)

EXAMPLE 219 N6-(3-Methoxyestra-1,3,5(10)-trien-17-yl)-N-t-boclysine,methyl ester

A 100 ml, 3-necked flask, equipped with a magnetic stirrer, was flamedried and then cooled in a nitrogen atmosphere. The flask was chargedwith 1.1 g of estrone 3-methyl ether dissolved in 20 ml oftetrahydrofuran and 30 ml of methanol. The solution was treated with 4 gof 3A molecular sieves followed by 1.51 g of α t-boc lysine. The pH ofthe slurry was adjusted to 6.0 with glacial acetic acid. The slurry wasthen treated with 243 mg of sodium cyanoborohydride and the reactionmixture refluxed for 18 hours. The reaction mixture was filtered throughCelite and the solids washed with 200 ml of methanol/tetrahydrofuran(4:3). The filtrate was concentrated in vacuo.

The crude product (4.6 g) was chromatographed on 285 g of 234-400 meshsilica gel. The column was packed and eluted with 95:4.5:0.5chloroform/methanol/concentrated ammonium hydroxide. An initial fractionof 500 ml was collected followed by 6 ml fractions.

Based on their TLC homogeneity, fractions 1-70 were combined affording1.1 g of title compound. NMR (CDCl₃, TMS) δ 7.3-7.1, 6.8-6.5, 5.25-5.0,4.5-4.05, 3.8, 3.75 3.01-1.0, 1.5 and 0.75 ppm.

EXAMPLE 220 N6-(3-Methoxyestra-1,3,5(10)-trien-17-yl), L-lysine, methylester

A 10 ml, 2-necked flask, equipped with a magnetic stirrer, was flamedried and then cooled in a nitrogen atmosphere. The flask was chargedwith 50 mg of the title compound from Example 219 dissolved in 5 ml ofmethylene chloride. The solution was cooled to 0° C. The solution wasthen treated with 2.6 ml of trifluoroacetic acid and stirred at roomtemperature for 2 hours. The reaction mixture was concentrated in vacuo.

The crude product (89 mg) was chromatographed on 7 g of 70-230 meshsilica gel. The column was packed and eluted with (89:10:1)chloroform/methanol/concentrated ammonium hydroxide. An initial fractionof 5 ml was collected followed by 1 ml fractions. Based on their TLChomogenity, fractions 16-24 were combined affording 31 mg of the titlecompound. NMR (CDCl₃, TMS): δ 7.25-7.1, 6.75-6.5, 3.75, 3.7, 3.0-1.0 and0.7 ppm.

EXAMPLE 221 1H-Pyrrole-1-acetic acid,2,5-dihydro-α[4-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]butyl]-2,5-dioxo-,methyl ester

A 25 ml, 2-necked flask, equipped with a magnetic stirrer, was flamedried and then cooled in a nitrogen atmosphere. The flask was chargedwith 163 mg of the title compound from Example 220 slurried in 20 ml ofo-xylene. The solution was treated with 45 mg of maleic anhydride, andthe reaction mixture was refluxed for 2 hours. The reaction mixture wasallowed to cool and then concentrated in vacuo.

The crude product (143 mg) was chromatographed on 25 g of 230-400 meshsilica gel. The column was packed and eluted with (95.2:4-3:0.5)chloroform/2-propanol/concentrated ammonium hydroxide. An initialfraction of 40 ml was collected followed by 2 ml fractions. Based ontheir TLC homogeneity, fractions 9-20 were combined affording 64 mg ofthe title compound. NMR (CDCl₃, TMS): δ 7.≅14 7.0, 6.8-6.5, 4.8-4.5,3.75, 3.7, 3.0-1.0 and 0.75 ppm. (PLA2)

EXAMPLE 222 1H-Pyrrole-2,5-dione,1-[3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]propyl].

A 25 ml, 2-necked flask, equipped with a magnetic stirrer, was flamedried and then cooled in a nitrogen atmosphere. The flask was chargedwith 246 mg of the first title compound from Example 68, slurried in 15ml of o-xylene. The solution was treated with 106 ml of maleicanhydride, and the reaction mixture was refluxed for 45 minutes and thenconcentrated in vacuo.

The crude product (360 mg) was chromatographed on 30 g of 230-400 meshsilica gel. The column was packed and eluted with (95:4.5:-0.5)chloroform/methanol/concentrated ammonium hydroxide. An initial fractionof 140 ml was collected followed by 5 ml fractions. Based on their TLChomogeneity, fractions 1-130 were combined affording 98 mg of impuretitle compound. The 98 mg of impure title compound was rechromatographedon 25 g of 230-400 mesh silica gel. The column was packed and elutedwith (96.4:3.2:0.4) chloroform/2-propanol/concentrated ammoniumhydroxide. Based on their TLC homogeneity, fractions 19-45 were combinedaffording 48 mg of title compound, m.p. 105-106. (PLA2)

EXAMPLE 223 1,6-Hexanediamine, N-estra-1,3,5(10)-trien-17-yl

1,6-Hexanediamine (1.2 g) was dissolved in 20 ml of methanol and the pHof the solution was lowered to 6.6 with glacial acetic acid. To thissolution was added 500 mg of estra-1,3,5-(10)-trien-17-one) and 20 ml ofTHF. Then 166 mg of sodium cyanoborohydride was added and the reactionwas brought to reflux. After 7 hours TLC (CHCl₃ /10% NH₄ OH in MeOH(9:1)) revealed product formation with no starting material present. Thesolution was made basic with concentrated NH₄ OH and dried byevaporation to produce 2.3 g of crude product. The entire crude waschromatographed on 100 mg of 230-400 mesh silica gel, eluting with CHCl₃/10% NH₄ OH in MeOH (9:1). After 150 ml was collected, 7 ml fractionswere taken. Fraction numbers 32-90 yielded 492 mg of the title compound.MS C₂₄ H₃₉ N₂ Meas. 355.3082. (PLA2 )

EXAMPLE 224 1,3-Propanediamine,N-[(17β)-3-[3-(dimethylamino)propoxy]estra-1,3,5(10)-trien-17-yl]-N,N',N'-trimethyl.

A flame dried 50 ml 2-necked round-bottomed flask, equipped with anitrogen inlet tube, 10 ml constant pressure addition funnel, andmagnetic stir bar, was charged with a 60% dispersion of sodium hydride(0.113 g). The dispersion was washed with hexane (3X), the hexane beingremoved by suction through a gas dispersion tube. The sodium hydride wascovered with 5 ml of dimethylformamide and the title compound fromPreparaion 11 (0.338 g) in 10 ml of dimethylformamide was added over 15minutes. After stirring for 1 hour at room temperature, the solidazetidine salt (0.307 g) from Preparation 10 was added. After anadditional 31/2 hours, another portion of the azetidine salt (50 mg) wasadded and stirring continued overnight. The reaction contents werepoured into ice water and the aqueous solution was extracted wihtchloroform (3X). The combined organic layers were washed with brine,dried (MgSO₄), filtered, and concentrated under high vacuum to give0.416 g of a semi-solid.

The semi-solid was chromatographed on 60 g of 230-400 mesh silica geleluting with chloroform containing 4-6% of a methanol/ammonium hydroxide(9/1) mixture. An initial fraction of 90 ml was collected followed by 5ml fractions. Fractions 137, 138, and 150-170 contained impure productby TLC and were rechromatographed on the same column eluting withchloroform containing 6% of a methanol/ammonium hydroxide (9/1) mixture.An initial fraction of 100 ml was collected followed by 5 ml fractions.Fractions 5-15 were nearly homogeneous by TLC and were combined withfractions 139-149 from the first chromatography to give 0.253 g of acolorless oil. IR: νmax (neat) 1610, 1501, 1461, 1382, 1313, 1282, 1256,1237,1179, 1155, 1061, 1042, 1011, and 841 cm⁻¹. (PLA2)

EXAMPLE 225 17β-t-Butyldimethylsilyoxy-5α-estran-3-one

10 g of 17β-hdyroxy-5α-estran-3-one was dissolved in 150 ml of DMF. 3.7g of imidazole were then added and the mixture was cooled to 0° C. 6.5 gof t-butyl dimethyl silyl chloride were added and the mixture wasstirred and gradually warmed to room temperature followed by stirringfor two days. TLC (hexane/EtOAc (9:1)) revealed that the reaction wascomplete. The reaction mixture was diluted with water and then extractedwith 500 ml of hexane/EtOAc (9:1) (2X). The combined organic layers werewashed with brine, dried over MgSO₄, filtered, and evaporated underreduced pressure to produce crude product (14.6 g). The entire crudeyield was field chromatographed with the Aldrich flash chromatographysystem. The 600 ml column was filled with 7 inches of 230-400 meshsilica gel, eluting with hexane/EtOAc (10:1). Once the sample wasloaded, 30 ml fractions were taken. Fraction numbers 4-13, yielded 14 gof the title compound, m.p. 103°-104° C.

EXAMPLE 226 17β-t-Butyldimethylsilyloxy-5α-estran-3α(and 3β)-ol

14 g of the title compound from Example 225 was dissolved in 100 ml ofCH₂ Cl₂, diluted with methanol (750 ml) and cooled to 0° C. 7.0 g ofNaBH₄ was added in portions until TLC (hexane/EtOAc (9:1)) no longerrevealed starting material. Excess NaBH₄ was quenched by adding 2MNaHSO₄ until gassing ceased (pH of 4). The mixture was diluted withwater and EtOAc and the organic layer was separated. The aqueous layerwas extracted with EtOAc and the combined extracts were then washed withwater and brine, dried over MgSO₄, filtered, and evaporated underreduced pressure to yield crude product (15.3 g). The entire crude waschromatographed on 1000 g of 230-400 mesh silica gel eluting with CH₂Cl₂ /acetone (98:2). After 1200 ml of column volume was collected, 17 mlfractions were taken. Fraction numbers 130-210 yielded 3.2 g of theα-isomer; m.p. 160°-162° C. Fractions numbers 280-430 yielded 10.9 g ofthe β-isomer, m.p. 135°-138° C.

EXAMPLE 227 17β-t-Butyldimethylsilyloxy-5α-estran-3β-ol,3-(2-cyanoethyl)ether

5.1 g of title compound from Example 226 was dissolved in 100 ml ofbenzene (stored over 4 Å sieves). 1.7 ml of acrylonitrile (purifiedaccording to "Purification of Laboratory Chemicals," D. D. Perrin etal., pp. 87-88) was then added followed by 0.24 ml of Triton B and themixture was stirred at room temperature. TLC (hexane/EtOAc (5:1))revealed product formation, approximately 70-80% complete. The crudeproduct was diluted with EtOAc, washed with water and brine, dried overMgSO₄, filtered, and evaporated under reduced pressure to produce crudeproduct (5.82 g). The entire crude was chromatographed on 430 g 70-230mesh silica gel, eluting with hexane/EtOAc (5:1). After 500 ml wascollected, 18 ml fractions were collected. Fractions 45-90 yield 4.43 gof the title compound. MS C₂₇ H₄₈ NO₂ S: Theory 446.3454, Measured446.3480.

EXAMPLE 228 3β,17β-Dihydroxy-5α-estrane, 3-(2-cyanoethyl)ether (3α-)-

4.4 g of the cyano-ether steroid from Example 227 was dissolved in 10 mlof CH₂ Cl₂ and then diluted with 200 ml of MeOH. 40 ml of CH₂ Cl₂ wasthen added and the reaction mixture became slightly turbid. 32 ml of amethanolic 3.2M HCl solution (final reaction concentration 0.4M) wasthen added. After 20 minutes, TLC (hexane/EtOAc (3:1)) revealed that thereaction was complete. The product was then diluted with water andEtOAc, washed again with water and then with brine, dried over MgSO₄,filtered and evaporated unde reduced pressure to produce crude product(3.73 g). The entire crude was flash chromatographed on 8 inches of230-400 mesh silica gel, eluting with CH₂ Cl₂ /acetone (98:2),collecting 25 ml fractions. Fraction numbers 8-25, yield 3.2 g of titlecompound. MS [M⁺ ] 331 found 331.

EXAMPLE 229 3β-Hydroxy-5α-estran-17-one, 3-(2-cyanoethyl)ether The samefor the (3α)-isomer

1.0 g of the cyano ehter steroid from Example 228 was dissolved in 20 mlof acetone and then cooled to 0° C. 1.0 ml of Jones Reagent was thenadded and this was stirred at 0° C. for 45 minutes. TLC (CH₂ Cl₂/acetone (98:2)) showed no starting material and the reaction wasquenched with approximately 5 ml of 2-PrOH. The reaction mixture wasthen diluted with water and EtOAc. The organic layer was separated andwashed with water and brine, dried over MgSO₄, filtered and evaporatedunder reduced pressure to produce crude product (1.1 g). MS C₂₁ H₃₁ NO₂: Theory 329.2355, Measured 329.2342.

EXAMPLE 230 Propanenitrile,3-[[3β,5α)-17-[[6-aminohexyl)amino]estran-3yl]oxy]

1.78 g of 1.6 -Hexanediamine was dissolved in 30 ml of MeOH and the pHof the solution was adjusted to 6.0 with glacial acetic acid. To thissolution was added 990 mg of the α cyano ether ketone from Example 229and 30 ml of THF, followed by 251 mg of sodium cyanoborohydride. Thereaction mixture was then refluxed overnight. TLC (CHCl₃ /10% NH₄ OH inMeOH (4:1)) revealed product formation with a trace of starting materialremaining. The entire reaction mixture was worked by evaporating underreduced pressure to produce crude product (7.98 g). The entire crude waschromatographed on 285 g of 230-400 mesh silica gel, eluting with CHCl₃/10% NH₄ OH in MeOH (4:1). After 425 ml was collected, 11 ml fractionswere taken. Fraction numbers 20-140 yielded 1.05 gf of product. MS C₂₇H₄₈ N₃ O: Theory 430.3786, Measured 430.3797. (PLA2)

EXAMPLE 231 Propanenitrile,3-[[(3α,5α)-17-[(6-aminohexyl)amino]estran-3-yl]oxy]

Following the procedure of Example 230 but starting with the α-cyanoether ketone, the title compound is produced. MS C₂₇ H₄₈ N₃ O: Theory430.3797, Measured 430.3786. (PLA2)

EXAMPLE 232 1,6-Hexanediamine,N-[(3β,5α)-3-(3-aminopropoxy)estran-17-yl]

106 mg of lithium aluminum hydride was slurried in 25 ml of ether, and asolution containing 300 mg of the cyano ether steriod from Example 230in 45 ml of ether was added in a dropwise manner. The solution was thenrefluxed for 2 hours. TLC (CHCl₃ /15% NH₄ OH in MeOH (4:1)) revealedproduct formation. The reaction was quenched with 212 ml of water and170 ml of a 10% NaOH solution and was stirred overnight. The nextmorning the white solid was filtered and then washed with hot CHCl₃. Thefiltrate was then evaporated under reduced pressure to yield crudeproduct (275 mg). The entire crude was chromatographed on 60 mg of230-400 mesh silica gel, eluting at first with CHCl₃ /4.2M NH₃ in MeOH(95:5) for 5 column volumes (375 ml) then followed by CHCl₃ /4.2M NH₃ inMeOH (9:1). Three ml fractions were then taken. Fraction numbers 141-230yielded 216 mg of product. MS C₂₇ H₅₂ N₃ O: Theory 434.4110, Measured434.4152. (PLA2)

EXAMPLE 233 1,6-Hexanediamine,N-[(3α,5α)-3-(3-aminopropoxy)estran-17-yl]-

Following the procedure of Example 232, but starting with the cyanoether steroid of Example 231, the title compound is produced. MS C₂₇ H₅₂N₃ O: Theory 434.4110, Measured 434.4144. (PLA2)

EXAMPLE 234 1,3-Propanediamine,N'-[(3β,5α)-3-2-cyanoethoxy)estran-17-yl)-N,N-dimethyl-

460 mg of N,N-Dimethylpropyldiamine was dissolved in 10 ml of MeOH andthe pH of the solution was adjusted to 6.0 with glacial acetic acid. Tothis solution was added 297 mg of the α or β-cyano ether steroid fromExample 229 and 10 ml of THF, followed by 75 mg of sodiumcyanobvorohydride. The reaction mixture was refluxed overnight. TLC(CHCl₃ /4.2M NH₃ in MeOH (12:1)) revealed product formation with a traceof starting material remaining. The reaction mixture was evaporatedunder reduced pressure to produce crude product (2.88 g). The entirecrude was chromatographed on 100 g of 230-400 mesh silica gel, elutingfirst with CHCl₃ /4.2M NH₃ in MeOH (15:1) for 13/4 column volumes (250ml), then followed by CHCl₃ /4.2M NH₃ in MeOH (15:1). Five ml fractionswere then taken. Fraction numbers 3-50 yielded 173 mg of product. MS [M⁺] 415, Found 415.

EXAMPLE 235 1,3-Propanediamine,N'-[(3β,5α)-3-2-cyanoethoxy)estran-17-yl)-N,N-dimethyl]-N,N,N'-trimethyl-

170 mg of the α or β-cyano ether steroid from Example 234 was dissolvedin 2 ml of acetonitrile and the pH was lowered to 6.5 with glacialacetic acid. 0.16 ml of a 37% formaldehyde solution was then addedfollowed by 41 mg of sodium cyanoborohydride. The reaction mixture wasstirred overnight at room temperature. TLC (CHCl₃ /4.2M NH₃ in MeOHn12:1)) revealed product formation with a trace of starting materialpresent. The reaction mixture was evaporated under reduced pressure toproduce crude product (287 mg). The entire crude was chromatographed on25 g of 230-400 mesh silica gel, eluting with CHCl₃ /4.2M NH₃ in MeOH(17:1). After 55 ml of column volume was collected, 3 ml fractions weretaken. Fraction numbers 3-13 yielded 122 mg of product. MS [M⁺ ] 429.

EXAMPLE 236 1,3-Propanediamine, N-[(3β,5α)-3-(3-aminopropoxy)estran-17-yl]-N,N',N'-trimethyl-

42 mg of lithium aluminum hydride was slurried in 10 ml of dry ether anda solution containing 121 mg of the β-cyano ether steroid from Example236 was dissolved in 20 ml of ether. After the addition was completed,the mixture was refluxed for 2 hours. TLC (CHCl₃ /4.2M NH₃ in MeOH(12:1)) revealed product formation. The reaction mixture was quenchedwith 84 μl of water and 67 μl of 10% NaOH solution and stirredovernight. The precipitate formed was filtered and then washed severaltimes with hot CHCl₃. The combined filtrate was evaporated under reducedpressure to produce crude product (104 mg). The entire crude waschromatographed on 25 g of 230-400 mesh silica gel, eluting with CHCl₃/4.2M NH₃ in MeOH (15:1). After 50 ml of 1 column volume was collected,2 ml fractions were taken. Fraction numbers 23-90 yielded 97 mg ofproduct. MS C₂₇ H₅₂ N₃ O: Theory 434.4110, Measured 434.4144. (PLA2)

EXAMPLE 237 1,3-Propanediamine,N-[(3α,5α)-3-(3-aminopropoxy)estran-17-yl]-N,N'N'-trimethyl-

Following the procedure of Example 236, but starting with the α-cyanoether steroid of Example 235, the title compound is produced. MS C₂₇ H₅₂N₃ O: Theory 434.4110, Measured 434.4144.

EXAMPLE 238 1,3-Propanediamine,N'-[(3β,5α)-3-2-cyanoethoxy)estran-17-yl)-N,N-dimethyl-

100 mg of the β-cyano ether steroid from Example 229 and 153 mg of3-dimethylaminopropylamine were placed in a 10 ml 2-necked flask. 68 μlof 95% formic acid were then added and the rection mixture was heated to150° C. The reaction was heated at 130° C. for about 5 to 10 minutes andthe temperature gradually increased to 150° C. within 30 minutes. Thenthe reaction was heated an additional 1 hour. TLC (CHCl₃ /10% NH₄ OH inMeOH (7:1)) revealed product formation. The reaction mixture was removedfrom the oil bath, allowed to cool, diluted with CHCl₃, transferred andevaporated under reduced pressure to product crude product (253 mg). Theentire crude was chromatographed on 7 g of 230-400 mesh silica gel,first eluting with CHCl₃ /10% NH₄ OH in MeOH (10:1). After 10 ml ofcolumn volume was collected 1 ml fractions were taken. Fraction numbers14-25 yielded 103 mg (105A). Since the separation was poor the 105A wasrechromatographed on 7 g of 230-400 mesh silica gel, eluting with CHCl₃/10% NH₄ OH in MeOH (19:1). After 10 ml of column volume was collected 1ml fractions were taken. Fraction numbers 25-34 yielded 4/7 mg of impureproduct (105B).

The entire 47 mg of 105B was placed on 8 of 230-400 mesh silica gelcolumn, eluting with CHCl₃ /4.2M NH₃ in MeOH (99:1). After 10 ml ofcolumn volume was collected, 40-1 ml fractions were collected. Then 90-1ml fractions of CHCl₃ /4.2M NH₃ in MeOH (98:2) were collected. TLCanalysis revealed the upper Rf impurities were separated. Finally thesolvent polarity was increased to CHCl₃ /4.2M NH₃ in MeOH (97:3) andfractions were collected. Fraction numbers 185-245 yielded 19 mg ofproduct. MS [M⁺ ] 415, Found 415.

EXAMPLE 239 1,3-Propanediamine,N'-[(3β,5α)-3-(3-aminopropoxy)estran-17-yl]-N,N-dimethyl-

7 mg of lithium aluminum hydride was slurried in 2 ml of ether and asolution containing 19 mg of the cyano ether from Example 238 in 7 ml ofether was added. Once the addition was completed, the reaction mixturewas refluxed for 2 hours. TLC (CHCl₃ /5% NH₄ OH in MeOH (4:1)) revealedproduct formation with a trace of starting material. Therefore, 1.4 μlof water and 1.1 μl of a 10% NaOH solution were added and the reactionmixture was stirred overnight. The ether solution and white precipitatewere filtered and the precipitate was washed several times with hotCHCl₃. The filtrate was evaporated under reduced pressure to produce thecrude product (19.7 mg). The entire crude was chromatographed on 7 g of230-400 mesh silica gel, eluting with CHCl₃ /4.2M NH₃ in MeOH (95:5) for5 column volumes and then eluted with CHCl₃ /4.2M NH₃ in MeOH (9:1 )collecting 2 ml fractions. Fraction numbers 26-50 yielded 12 mg ofproduct. MS C₂₆ H₅₀ N₃ O: Theory 420.3954, Measured 420.3956. (PLA2)

EXAMPLE 240 1H-Pyrrole-1-acetamide,2,5-dihydro-N-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-2,5-dioxo-,monohydrochloride

100 mg of the title compound from Example 76 and 72 mg of the p-nitroester nitrophenyl succinimidoylacetate were dissolved in 3 ml of DMF andstirred at room temperature for 18 hours. TLC (CHCl/10% NH₄ OH in t-PrOH(10:1) revealed product formation. The reaction mixture was diluted withwater, extracted several times with CHCl₃, and the organic layer wasseparated, washed with saturated NaCl, dried over MgSO₄, filtered, andevaporated under reduced pressure (high vacuum) to produce crude product(130 mg). The entire crude was chromatographed on 25 g of 230-400 meshsilica gel eluting with CHCl₃ /10% Et₃ N in 2-PrOH (4:1). After 50 ml ofcolumn volume was collected, 2-3 ml fractions were taken. The entiremixture of components came out on the first column volume as well as thefirst 10 fractions. This was combined to yield 75 mg. Anotherchromatography was performed, the same procedure was followed with oneexception, the solvent system was CHCl₃ /10% Et₃ N in 2-PrOH (10:1),after 50 ml was collected, 2-3 ml fractions were taken. Fraction numbers45-55 yielded 47 mg of crude product. MS C₃₁ H₄₄ N₃ O₄ : Theory522.3332, Measured 522.3309.

The entire 47 mg of crude product was dissolved in a minimum amount ofCHCl₃ (approximately 1 ml). Then a 2.8M HCl in EtOAc solution (1 ml) wasadded with stirring. After 15 minutes at room temperature the reactionmixture was still clear, therefore, 15 ml of n-hexane was added and theresulting light yellow solid was isolated. Upon filtration it was notedthat the solid was tacky, therefore a nitrogen atmosphere was placedaround the filtration apparatus. The 47 mg was placed in the vacuumdesicator overnight at 0.1 torr and room temperature. The yield aftervacuum desication was 17 mg. (PLA2)

EXAMPLE 241 1H-Pyrrole-2,5-dione,1-[3-[(3-melthoxyestra-1,3,5(10)-trien-17-yl)oxy]propyl]

100 mg of the amine ether steroid3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)oxy]-1-propanamine and 34 mg ofmaleic anhydride were slurried in 10 ml of o-xylene and refluxed for 1hour. TLC (CHCl₃ /4.2M NH₃ in MeOH (12:1)) revealed all startingmaterial gone. After 2 hours total reflux the reaction was stopped andallowed to stand for 2 days. TLC (CHCl₃ /25% NH₄ OH in MeOH (4:1))revealed that the maleiamic acid was formed. The reaction mixture wasplaced on the high vacuum, evaporated under reduced pressure to producecrude product (120 mg).

The 120 mg of maleamic acid was slurried in 2 ml of xylene and 1 ml DMF,9 mg of Amberlyst 15 resin were added and the reaction mixture washeated to reflux. After 3 hours of reflux, TLC (CHCl₃ /4.2M NH₃ in MeOH(12:1)) revealed no product formation, therefore the mixture wasrefluxed overnight. TLC in CHCl₃ /25% NH₄ OH in MeOH (4:1) revealedproduct formation and that some of the maleamic acid was present. Thereaction was cooloed and then was evaporated under reduced pressureunder high vacuum to produce crude product (129 mg). The entire crudewas chromatographed on 25 g of 230-400 mesh silica gel eluting with 100%CHCl₃. After 50 ml (1 column volume) was collected, 2 ml fractions weretaken. Fraction numbers 3-9 yielded 10 mg of product. MS C₂₆ H₃₃ NO₃ :Theory 423.2409, Measured 423.2386. (PLA2)

EXAMPLE 242 Propanenitrile,3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)oxy]-

1.16 g of the 17β-alcohol steroid estradio, 3-melthyl ether wasdissolved in 20 ml of benzene (stored over 4 Å sieves). 0.53 mlacrylonitrile (purified according to "Purification of LaboratoryChemicals," D. D. Perrin et al. pp. 87-88) was then added followed by 73μl of Triton B and the mixture was stirred at room temperature for 2days. TLC (hexane/EtOAc (5:1)) revealed product formation with a traceof starting material present. The crude product was worked up bydiluting with EtOAc and brine. The aqueous layer was made acidic with 2MNaHSO₄ and the organic layer was separated, dried over MgSO₄, filtered,and evaporated under reduced pressure to produce crude product (1.51 g).The entire crude was chromatographed on 285 g of 230-400 mesh silicagel, eluting with CH₂ Cl₂ /hexane (2:1). After 500 ml was collected, 12ml fractions were taken. Fraction numbers 120-220 yielded 1.4 g ofproduct. MSM⁺ 339 found 339.

EXAMPLE 243 1-Propanamine,3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)oxy]-

626 mg of lithium aluminum hydride was slurried in 150 ml of dry etherand a solution containing 1.4 g of the cyano ether steroid from Example242 in 300 ml of dry ether. After the addition was completed the mixturewas refluxed for 2 hours. TLC (CHCl₃ /4.2M NH₃ in MeOH (12:1)) revealedproduct formation but starting material was stil present. An additional626 mg of LiAlH₄ was added. After 30 minutes TLC revealed about 75%completion. The reaction mixture was refluxed for 11/2 hours, afterwhich TLC indicated no starting material left. 2.5 ml of water was addedcarefully followed by 2.0 ml of a 10% sodium hydroxide solution. Themixture was stirred overnight. The reaction mixture was filtered and thesolids washed several times with hot CHCl₃. The combined organicfiltrate was then evaporated under reduced pressure to produce crudeproduct (1.53 g). The entire crude was chromatographed on 285 g of 230-400 mesh silica gel, eluting with CHCl₃ /4.2M NH₃ in MeOH (24:1). After475 ml (1 column volume) was collected, 12 ml fractions were taken.Fraction numbers 193-210 yielded 566 mg. MS C₂₂ H₃₄ NO₂ : Theory344.2589, Measured 344.2603.

EXAMPLE 244 Acetamide,2-bromo-N-[3-[(3-methoxyestra-1,3,5(10)-trien-17-yl)oxy]propyl]-

100 mg of the amino ether steroid from Example 243 was dissolved in 8 mlof THF and cooled to 0° C. Then a solution containing 65 mg of thesuccinimide N-(α-bromoacetoxy)-succinimide in 4 ml of THF was added. Themixture was stirred. After 1 hour at 0° C. the mixtue was graduallywarmed to room temperature. After 1-2 hour, TLC (CHCl₃ /4.2M NH₃ in MeOH(24:1)) revealed product formation with only a trace of startingmaterial present. The reaction mixture was evaporated under reducedpressure to produce crude product (167 mg). The entire crude waschromatographed on 25 g of 230-400 mesh silica gel, eluting with CHCl₃/4.2M NH₃ in MeOH (24:1). After 40 ml was collected (0.8 column volume),1.5-2.0 ml fractions were taken. Fraction numbers 3-9 yielded 102 mg ofproduct. MS C₂₄ H₃₅ BrNO₃ Theory 464.1801, Measured 464.1790.

EXAMPLE 245 17β-[(2-(4-Aminosulfonylphenyl)ethyl)aminoυ-5α-androstaneHemihydrate

A solution of 3.39 g of 4-(2-aminoethyl)benzenesulfonamide in 50 ml ofMeOH and 150 ml of THF was acidified with 3 ml (3.15 g) of acetic acid.Then 2.32 g of 5α-androstan-17-one was added. After a solution wasobtained, 1.2 g of sodium cyanoborohydride was added. The resultingsolution was stirred for 5 hours. An additional 1.2 g of sodiumcyanoborohydride was added. The stirring was continued for 19 hours. Thesolvent was evaporated from the solution. The residue was treated with200 ml of water, made basic with 50% NaOH solution, and extracted withCH₂ Cl₂ (3×100 ml). The combined extracts were washed with 50 ml ofbrine and dried over MgSO₄. Evaporation of the solvent left 3.75 g ofsolid. The solid was chromatographed on a 400 g column of silica gel.The column was eluted with 10% MeOH--CH₂ Cl₂ and 100 ml fractions werecollected. The fractions were assayed by silica gel TLC (1×4") (10%MeOH--CH₂ Cl₂). Fractions 18-30 were combined and crystallized fromMeOH--H₂ O giving 0.76 g of the title compound.

Some of the compounds of the present invention also are useful asangiogenic and angiostatic compounds, i.e., compounds that stimulate orinhibit growth of blood vessels respectively. These activities may beassayed by the methods described in J. Folkman, et al., Science, 221,pp. 719-25 (1983).

The preferred compounds for angiogenic use as assayed in the absence ofheparin include1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione,N-[3-(N'-morpholino)propyl]-3-methoxyestra-1,3,5(10)-trien-17β-amine,3-methoxy-N-(3'-pyridinylmethyl)estra-1,3,5(10)-trien-17β-amine,N-[3-(dimethylamino)propyl]-3-methoxy-N-methylestra-1,3,5(10)-trien-17.beta.-amine,N-[3'-(dimethylamino)propyl]-3-methoxy-N-methylestra-2,5(10)-dien-17β-amine,and1-[6-[(3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-3,4-dimethyl-1H-pyrrole-2,5-dione.

The preferred compounds for angiostatic use includeN-(5-fluoro-2,4-dinitrophenyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,6-hexanediamineandN-(2,4-dinitrophenyl)-N'-[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-1,6-hexanediamine.

The angiostatic compounds of the present invention are useful intreating the following diseases and injuries: head trauma, spinaltrauma, septic or traumatic shock, stroke, and hemorrhagic shock. Inaddition, utility in cancer as well as other disorders or physiologicalphenomena dependent on angiogenesis such as embryo implanation(antifertility), arthritis, and atherosclerosis is exhibited with thesecompounds optionally co-administered with oral heparin or systemicheparin fragments (see J. Folkman, et al., supra).

The angiostatic compounds can be administered orally, intramuscularly,intravenously and by suppository, and the effective dosage range is 10to 1500 mg/kg/day. The compounds of the present invention may becoadministered with low doses of glucocorticoids. For the treatment ofcancer including head tumors and other conditions dependent uponangiogensis a preferred dosage range of the angiostatic compounds is 50to 500 mg/kg-day for 30 days repeated for 30 additional days after a 30day respite or on a chronic intermittent basis such as every other daytherapy until tumor regression or absence of metastases is observed. Thepreferred route of administration is orally, by suppository orintramuscularly. For the treatment of arthritis the preferred dosagerange of the angiostatic compounds is 10 to 250 mg/kg/day or every otherday until absence or significant reduction in associated symptoms isobserved. For the treatment of atherosclerosis the preferred dosagerange of the angiostatic compounds is 10 to 250 mg/kg/day or every otherday chronically. And, for the disruption of or prevention of embryoimplantation the preferred dosage range of the angiostatic compounds is10 to 250 mg/kg-day chronically to fertile women. When coadministeringan angiostatic compound with heparin or a heparin fragment in practicingthe present invention the amount of heparin or heparin to be utilizedvaries from 1,000 to 50,000 units/kg/day with heparin being administeredorally and heparin fragments being administered subcutaneously, orally,intramuscularly or intravenously.

The utility of the compounds of the present invention can bedemonstrated in various test models as follows: For head trauma, miceare struck on the head with a standard weight which is dropped from aset height. They are then dosed subcutaneously with the test compound.After one hour the motor abilities of the mice are assessed. Active testcompounds promote improved motor activity relative to controls. Forspinal trauma, see E. D. Hall and J. M. Braughler, Surg. Neurol. 18,320-327 (1982) and J. Neurosurg. 56, 838-844 (1982). Septic (traumatic)shock is demonstrated in a rat model whereby test compound isadministered and protection of the rats from the lethal effects ofendotoxin is measured. For stroke, the carotid arteries of geribls areligated for a brief period after which test compound is administeredsubcutaneously. The behavior of the gerbils is observed after a recoveryperiod, and gerbils receiving test compound display a more normalbehavior after the recovery period. And for hermorrhagic shock, bypublished procedure used to evaluate gluccorticoids. The inhibition ofangiogenesis associated with tumor formation and prolification istypically evaluated in the chick embryo or rabbit cornea, e.g., asreported by J. Folkman, et al., spra.

Sterile aqueous solutions of the angiostatic compounds typically willcontain other components such as preservatives, anti-oxidants, chelatingagents, or other stabilizers. Suitable preservatives can include benzylalcohol, the parabens, benzalkonium chloride, or benzoic acid.Anti-oxidants sucha s sodium bisulfite, ascorbic acid, propyl3,4,5-trihydroxy benzoate, and the like may be employked. Chelatingagents such as citrate, tartrate, or ethylenediaminetetraacetic acid(EDTA) may be used. Other additives useful as stabilizers ofcorticosteroid prodrugs (e.g., creatinine, polysorbate 80, and the like)may be employed.

Sterile aqueous solutiosn of the angiostatic compounds can beadministered to the patient being treated, i.e., a warm blooded mammal,including humans, intramuscularly or intravenously or orally.Additionally conventional solid dosage forms of the antiostaticcompounds can be administered orally to the patient being treated. Forexample, capsules, pills, tablets or powders of the angiostaticcompounds can be formulated in unit dosage forms incorporatingconventional fillers, dispersants, preservatives and lubricants. Alsosuppositories providing a sustained release of an angiostatic compoundcan be formulated using conventional inert materials such asbiodegradable polymers or synthetic silicones.

Heparin fragment means any part of the heparin compound havingsubstantially the same type of anti--angiogenic activity as heparin.##STR15##

We claim:
 1. A compound selected from the group consisting ofN-(5-fluoro-2,4-dinitropheny)-N'-(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)-1,6-hexanediamine,N-(2,4-dinitrophenyl)-N'-(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)-1,6-hexanediamineand pharmacologically acceptable acceptable salts thereof.
 2. A compoundaccording to claim 1,N-(5-fluoro-2,4-dinitrophenyl)-N'-(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)-1,6-hexanediamine.3. A compound according to claim 1,N-(2,4-dinitrophenyl)-N'-(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)-1,6-hexanediamine.